Modified microorganism and methods of using same for producing 2-propanol and1-propanol and/or 1,2-propanediol

ABSTRACT

The present disclosure provides a non-naturally occurring microorganism comprising: one or more polynucleotides encoding one or more enzymes in a pathway that produces acetyl-CoA; one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of cytosolic acetyl-CoA to 2-propanol; one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to 1-propanol and/or 1,2-propanediol, wherein the microorganism has reduced levels of pyruvate decarboxylase enzymatic activity (e.g., the microorganism comprises a disruption of one or more enzymes that decarboxylate pyruvate and/or a disruption of one or more transcription factors of one or more enzymes that decarboxylate pyruvate), and wherein the microorganism is capable of growing on a C6 sugar as a sole carbon source under anaerobic conditions. Also provided are methods of using the disclosed non-naturally occurring microorganisms in methods for the coproduction of 2-propanol and 1-propanol and/or 1,2-propanediol.

BACKGROUND

1-propanol (n-propanol, CH₃CH₂CH₂OH, CAS 71-23-8) is a primary alcoholtypically manufactured by catalytic hydrogenation of propionaldehyde,which is generally synthesized in large scale from ethylene in anenergy-intensive multi-step industrial process. This process involvesuse of toxic chemicals such as carbon monoxide and hydrogen at highpressure (e.g., 10-100 ATM) and high temperature (up to 200° C.).1-propanol can be used as an intermediate for further organic reactionsor as a building block for polymers such as propylene. Propylene is achemical compound that is widely used to synthesize a wide range ofpetrochemical products. For instance, this olefin is the raw materialused for the production of polypropylene, its copolymers and otherchemicals such as acrylonitrile, acrylic acid, epichloridrine andacetone. Propylene is typically obtained in large quantity scales as abyproduct of catalytical or thermal oil cracking, or as a co-product ofethylene production from natural gas. (Propylene, Jamie G. Lacson, CEHMarketing Research Report-2004, Chemical Economics Handbook-SRIInternational). Propylene is polymerized to produce thermoplasticsresins for innumerous applications such as rigid or flexible packagingmaterials, blow molding and injection molding.

2-propanol (isopropyl alcohol, CH₃CH₃CHOH, CAS 67-63-0) is a secondaryalcohol and is a structural isomer of 1-propanol. 2-propanol istypically produced by the weak acid process in which propene gas isabsorbed in, and reacted with, 60% sulfuric acid and the resultingsulfates hydrolyzed in a single step process. Another major currentmanufacturing process is catalytic hydration of propylene with water.Hydration can be gas phase with a phosphoric acid catalyst, mixed phasewith a cation-exchange resin catalyst or liquid phase using a tungstencatalyst. 2-propanol is used as an industrial solvent, a component ofindustrial and consumer products and as a disinfectant. Most 2-propanolgoes into the solvent market either directly or via conversion toacetone or one of acetone's derivatives—methyl isobutyl ketone, methylisobutyl carbinol, diacetone alcohol, or isophorone. 2-propanol's majorsolvent uses include inks, coatings, cosmetics and pharmaceuticals.

1,2-propanediol (propylene glycol, HO—CH₂—CHOH—CH₃, CAS 57-55-6) is anorganic compound with formula C₃H₈O₂. Industrially, propylene glycol isproduced from propylene oxide. Propylene glycol may be manufacturedusing either a non-catalytic high-temperature process at 200° C. (392°F.) to 220° C. (428 F), or a catalytic method, which proceeds at 150° C.(302° F.) to 180° C. (356° F.) in the presencefdon exchange resin or asmall amount of sulfuric acid or alkali. Propylene glycol can be used asa solvent, nontoxic antifreeze and to produce polyesteres compounds.

Given the world-wide demand for 2-propanol, 1-propanol, and1,2-propanediol, there exits a need in the art for improved methods fortheir production that overcome their current production drawbacksincluding the use of toxic and/or expensive catalysts, and highlyflammable and/or gaseous carbon sources.

SUMMARY

The present disclosure provides a non-naturally occurring microorganismcomprising: one or more exogenous polynucleotides encoding one or moreenzymes in a pathway that produces cytosolic acetyl-CoA (i.e.,acetyl-CoA is produced in the cytosol of the microorganism), wherein themicroorganism has reduced levels of pyruvate decarboxylase enzymaticactivity, and wherein the microorganism is capable of growing on a C6sugar as a sole carbon source and under anaerobic conditions.

The present disclosure covers the co-production of 1,2-propanol or1-propanol and 2-propanol in a eukaryote cell, such as a yeast, withreduced levels of pyruvate decarboxylase enzymatic activity, wherein themicroorganism has its native ethanol production shut-off, and whereinthe microorganism is capable of growing on a C6 sugar as a sole carbonsource under anaerobic or microaerobic conditions.

In order to eliminate the ethanol production in yeast it is necessary toknock out the activity of pyruvate decarboxylase, the enzyme thatdecarboxylates pyruvate making acetaldehyde and carbon dioxide. Inyeast, this enzyme comes in three isoforms, and its activity can becompletely knocked out by deleting the genes PDC1, PDC5 and PDC6. As aconsequence, the microorganism can not grow on C6 sugars as a solecarbon source such as glucose and consequently it is necessary to alterthe ability of the microorganism to import glucose, for example, bytruncating a transcription factor of the glucose importer MTH1. Also,the elimination of the pyruvate decarboxylase activity in the cell'scytoplasm renders the microorganism unable to grow under anaerobicconditions due to two factors: (1) the lack of an alternative route forcytoplasmic acetyl-CoA production, due to the lack of acetaldehyde thatwould be converted to acetate and acetyl-CoA; and (2) a redox imbalancedue to excess NADH because the NADH is no longer oxidized in theconversion of acetaldehyde to ethanol. Accordingly, the presentdisclosure discloses a series of complex deletions/truncations and geneintegrations that enables a new acetil-CoA overproducing yeast chassisfor the co-production of 1,2-propanol or 1-propanol and 2-propanol.

The present disclosure also provides a non-naturally occurringmicroorganism comprising: a disruption of one or more enzymes thatdecarboxylate pyruvate and/or a disruption of one or more transcriptionfactors of one or more enzymes that decarboxylate pyruvate; a geneticmodification that substantially decreases glucose import into themicroorganism; one or more polynucleotides encoding one or more enzymesin a pathway that produces cytosolic acetyl-CoA; one or morepolynucleotides encoding one or more enzymes in a pathway that catalyzea conversion of cytosolic acetyl-CoA to 2-propanol; and one or morepolynucleotides encoding one or more enzymes in a pathway that catalyzea conversion of dihydroxyacetone-phosphate to 1-propanol and/or1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more enzymes thatdecarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more enzymes that decarboxylate pyruvate includepdc1, pdc 5, and/or pdc6, and wherein the one or more transcriptionfactors of the one or more enzymes that decarboxylate pyruvate includepdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises an exogenous polynucleotidethat encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises a genetic modification in anendogenous polynucleotide that encodes a transcription factor involvedin glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the genetic modification is a truncation of the MTH1transcription factor.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more exogenous polynucleotides encoding one ormore enzymes in a pathway that produces acetyl-CoA encode i.) pyruvateformate lyase and pyruvate formate lyase activating enzyme, ii) pyruvatedehydrogenase, dihydrolipoyl transacetylase and dihydrolipoamidedehydrogenase, iii) pyruvate dehydrogenase, dihydrolipoyltransacetylase, dihydrolipoamide dehydrogenase, and pyruvatedehydrogenase complex protein X, or any combination thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism is a eukaryote selected from the groupconsisting of: yeast, filamentous fungi, protozoa, and algae.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetyl-CoA to acetoacetate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1-propanol and/or 1,2-propanediol include: one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofdihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofmethylglyoxal to hydroxyacetone, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of hydroxyacetone to1,2-propanediol, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to lactaldehyde, oneor more polynucleotides coding for enzymes in a pathway that catalyze aconversion of lactaldehyde to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of 1,2-propanediol to propionaldehyde, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of propionaldehyde to 1-propanol.

The present disclosure also provides a non-naturally occurringmicroorganism comprising: one or more exogenous polynucleotides encodingone or more enzymes in a pathway that produces cytosolic acetyl-CoA; oneor more polynucleotides coding for enzymes that catalyze a conversion ofcytosolic acetyl-CoA to 2-propanol; and one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofdihydroxyacetone-phosphate to 1-propanol and/or 1,2-propanediol, whereinthe microorganism has reduced levels of pyruvate decarboxylase enzymaticactivity, and wherein the microorganism is capable of growing on a C6sugar as a sole carbon source under anaerobic conditions.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate or a disruption in one or more polynucleotides that code for atranscription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more enzymes thatdecarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more enzymes that decarboxylate pyruvate includepdc1, pdc 5, and/or pdc6, and wherein the one or more transcriptionfactors of the one or more enzymes that decarboxylate pyruvate includepdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetyl-CoA to acetoacetate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1-propanol include and/or 1,2-propanediol: one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofdihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofmethylglyoxal to hydroxyacetone, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of hydroxyacetone to1,2-propanediol, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to lactaldehyde, oneor more polynucleotides coding for enzymes in a pathway that catalyze aconversion of lactaldehyde to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of 1,2-propanediol to propionaldehyde, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of propionaldehyde to 1-propanol.

The present disclsoure also provides methods for co-producing 2-propanolwith 1-propanol and/or 1,2-propanediol from a fermentable carbon sourceunder anaerobic conditions, the method comprising: providing afermentable carbon source; contacting the fermentable carbon source withthe non-naturally occurring microorganism as disclosed herein in afermentation media under substantially anaerobic conditions, andexpressing the polynucleotides in the microorganism for theco-production of 2-propanol with 1-propanol and/or 1,2-propanediol,wherein the microorganism co-produces 2-propanol with 1-propanol and/or1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the fermentable carbon source is sugarcane juice, sugarcanemolasses, hydrolyzed starch, hydrolyzed lignocellulosic materials,glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, orglycerol in any form or mixture thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the fermentable carbon source is a monosaccharide,oligosaccharide, or polysaccharide.

In some embodiments of each or any of the above or below mentionedembodiments, the produced 2-propanol with 1-propanol and/or1,2-propanediol are secreted by the microorganism into the fermentationmedia.

In some embodiments of each or any of the above or below mentionedembodiments, the methods further comprise recovering the produced2-propanol with 1-propanol and/or 1,2-propanediol from the fermentationmedia.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has no detectable pyruvate decarboxylaseenzymatic activity.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or morepolynucleotides that code for a transcription factor of an enzyme thatdecarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in each of the one ormore polynucleotides that code for enzymes that decarboxylate pyruvateor a disruption in each of the polynucleotides that code for atranscription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more polynucleotides is adeletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate code for pdc1, pdc5, and/or pdc6. In someembodiments of each or any of the above or below mentioned embodiments,the one or more polynucleotides that code for a transcription factor ofone or more enzymes that decarboxylates pyruvate code for pdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises an exogenous polynucleotidethat encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises a genetic modification in anendogenous polynucleotide that encodes a transcription factor involvedin glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the genetic modification is a truncation of the MTH1transcription factor. In an embodiment, the MTH1 transcription factormay have the amino acid sequence as set forth in SEQ ID NO: 1 and thetruncated MTH1 transcription factor may have the amino acid sequence setforth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the truncated MTH1 transcription factor has a longerhalf-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more exogenous polynucleotides encoding one ormore enzymes in a pathway that produces cytosolic acetyl-CoA encode i.)pyruvate formate lyase and pyruvate formate lyase activating enzyme, ii)pyruvate dehydrogenase, dihydrolipoyl transacetylase anddihydrolipoamide dehydrogenase, iii) pyruvate dehydrogenase,dihydrolipoyl transacetylase, dihydrolipoamide dehydrogenase, andpyruvate dehydrogenase complex protein X, or any combination thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism is a eukaryote.

In some embodiments of each or any of the above or below mentionedembodiments, the eukaryote is a yeast, filamentous fungi, protozoa, oralgae.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides coding for an acetoacetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentionedembodiments, the acetoacetyl-CoA hydrolase is produced by introducing amutation into the polynucleotide that encodes acetoacetyl-CoA:acetatetransferase. In some embodiments of each or any of the above or belowmentioned embodiments, the mutation is a E51D Glu-Asp mutationcorresponding to the numbering of SEQ ID NO: 3.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more exogenouspolynucleotides encoding one or more enzymes in pathways for theco-production of 1,2-propanediol and 2-propanol from a fermentablecarbon source under anaerobic conditions.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of 1,2-propanediol to propanaldehyde.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme is a B12-independent dehydratase.

In some embodiments of each or any of the above or below mentionedembodiments, the B12-independent dehydratase is from Clostridiumacetobutylicum, Clostridium glycolicum, Clostridium butyricum orRoseburia inulinivorans.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more exogenouspolynucleotides encoding one or more enzymes in pathways for theco-production of 2-propanol, 1, propanol and/or 1,2-propanediol from afermentable carbon source under anaerobic conditions.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofacetyl-CoA to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetyl-CoA to acetoacetate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofdihydroxyacetone-phosphate to 1,2-propanediol

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1,2-propanediol include: one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofdihydroxyacetone-phosphate to 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1-propanol include: one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion of pyruvateto 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of pyruvate to 1,2-propanediolinclude: one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of pyruvate to lactate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactate to lactoyl-CoA, one or more polynucleotides codingfor enzymes in a pathway that catalyze a conversion of lactoyl-CoA tolactaldehyde and/or one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion of pyruvateto 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of pyruvate to 1-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of pyruvate to lactate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactate to lactoyl-CoA, one or more polynucleotides codingfor enzymes in a pathway that catalyze a conversion of lactoyl-CoA tolactaldehyde, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of lactaldehyde to 1,2-propanediol,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of 1,2-propanediol to propionaldehyde, and/or oneor more polynucleotides coding for enzymes in a pathway that catalyze aconversion of propionaldehyde to 1-propanol.

The present disclosure also provides a non-naturally occurringmicroorganism comprising: a disruption of one or more enzymes thatdecarboxylate pyruvate and/or a transcription factor of an enzyme thatdecarboxylates pyruvate; a genetic modification that decreases glucoseimport into the microorganism; and one or more exogenous polynucleotidesencoding one or more enzymes in a pathway that produces cytosolicacetyl-CoA.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in one or more enzymes that decarboxylatepyruvate and/or a transcription factor of an enzyme that decarboxylatespyruvate results in reduced levels of pyruvate decarboxylase enzymaticactivity or no detectable pyruvate decarboxylase enzymatic activity.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more enzymes thatdecarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more enzymes that decarboxylate pyruvate includepdc 1, pdc 5, and/or pdc 6. In some embodiments of each or any of theabove or below mentioned embodiments, the transcription factor of anenzyme that decarboxylates pyruvate includes pdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises an exogenous polynucleotidethat encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises a genetic modification in anendogenous polynucleotide that encodes a transcription factor involvedin glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the genetic modification is a truncation of the MTH1transcription factor. In an embodiment, the MTH1 transcription factormay have the amino acid sequence as set forth in SEQ ID NO: 1 and thetruncated MTH1 transcription factor may have the amino acid sequence setforth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the truncated MTH1 transcription factor has a longerhalf-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more exogenous polynucleotides encoding one ormore enzymes in a pathway that produces cytosolic acetyl-CoA encode i.)pyruvate formate lyase and pyruvate formate lyase activating enzyme, ii)pyruvate dehydrogenase, dihydrolipoyl transacetylase anddihydrolipoamide dehydrogenase, iii) pyruvate dehydrogenase,dihydrolipoyl transacetylase, dihydrolipoamide dehydrogenase, andpyruvate dehydrogenase complex protein X, or any combination thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism is a eukaryote.

In some embodiments of each or any of the above or below mentionedembodiments, the eukaryote is a yeast, filamentous fungi, protozoa, oralgae.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides coding for an acetoacetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentionedembodiments, the acetoacetyl-CoA hydrolase is produced by introducing amutation into the polynucleotide that encodes acetoacetyl-CoA:acetatetransferase. In some embodiments of each or any of the above or belowmentioned embodiments, the mutation is a E51D Glu-Asp mutationcorresponding to the numbering of SEQ ID NO: 3.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more exogenouspolynucleotides encoding one or more enzymes in pathways for theco-production of 1,2-propanediol or 1-propanol and 2-propanol from afermentable carbon source under anaerobic conditions.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofacetyl-CoA to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetyl-CoA to acetoacetate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofdihydroxyacetone-phosphate to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1,2-propanediol include: one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion of pyruvateto 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of pyruvate to 1,2-propanediolinclude: one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of pyruvate to lactate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactate to lactoyl-CoA, one or more polynucleotides codingfor enzymes in a pathway that catalyze a conversion of lactoyl-CoA tolactaldehyde ando/or one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of 1,2-propanediol to propanaldehyde.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme is a B12-independent dehydratase.

In some embodiments of each or any of the above or below mentionedembodiments, the B12-independent dehydratase is from Clostridiumbutyricum, or Roseburia inulinivorans.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofdihydroxyacetone-phosphate to 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1-propanol include: one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion of pyruvateto 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of pyruvate to 1-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of pyruvate to lactate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactate to lactoyl-CoA, one or more polynucleotides codingfor enzymes in a pathway that catalyze a conversion of lactoyl-CoA tolactaldehyde, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of lactaldehyde to 1,2-propanediol,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of 1,2-propanediol to propionaldehyde, and/or oneor more polynucleotides coding for enzymes in a pathway that catalyze aconversion of propionaldehyde to 1-propanol.

The present disclosure also provides a non-naturally occurringmicroorganism comprising: one or more exogenous polynucleotides encodingone or more enzymes in a pathway that produces cytosolic acetyl-CoA; oneor more polynucleotides coding for enzymes that produce 1,2-propanediol,and wherein the microorganism has reduced levels of pyruvatedecarboxylase enzymatic activity, and wherein the microorganism iscapable of growing on a C6 sugar as a sole carbon source and underanaerobic conditions.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides encoding one or more enzymes in a pathway that producesacetate.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides encoding an acetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides encoding a phosphate acetyltransferase andacetyl-phosphate kinase.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides encoding a lactate CoA-transferase.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion of1,2-propanediol to 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of 1,2-propanediol to 1-propanolinclude: one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of 1,2-propanediol to propionaldehyde, and/orone or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of propionaldehyde to 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has no detectable pyruvate decarboxylaseenzymatic activity.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or morepolynucleotides that code for a transcription factor of an enzyme thatdecarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more polynucleotides is adeletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate code for pdc1, pdc5, and/or pdc6. In someembodiments of each or any of the above or below mentioned embodiments,the one or more polynucleotides that code for a transcription factor ofone or more enzymes that decarboxylates pyruvate code for pdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises an exogenous polynucleotidethat encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises a genetic modification in anendogenous polynucleotide that encodes a transcription factor involvedin glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the genetic modification is a truncation of the MTH1transcription factor. In an embodiment, the MTH1 transcription factormay have the amino acid sequence as set forth in SEQ ID NO: 1 and thetruncated MTH1 transcription factor may have the amino acid sequence setforth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the truncated MTH1 transcription factor has a longerhalf-life than an untruncated MTH1 transcription factor.

The present disclosure also provides a non-naturally occurringmicroorganism comprising: one or more exogenous polynucleotides encodingone or more enzymes in a pathway that produces cytosolic acetyl-CoA; oneor more polynucleotides coding for an acetyl-CoA acetyltransferase; oneor more polynucleotides coding for enzymes that produce 1,2-propanediol,wherein the microorganism has reduced levels of pyruvate decarboxylaseenzymatic activity, and wherein the microorganism is capable of growingon a C6 sugar as a sole carbon source and under anaerobic conditions.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides encoding one or more enzymes in a pathway that produces1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides coding for an acetoacetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides encoding one or more enzymes in a pathway that produces2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism further comprises one or morepolynucleotides coding for a HMG-CoA synthase and HMG-CoA lyase (see,e.g., WO2014076232).

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has no detectable pyruvate decarboxylaseenzymatic activity.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in each of the one ormore polynucleotides that code for enzymes that decarboxylate pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more polynucleotides is adeletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides code for pyruvatedecarboxylase 1, 5, and/or 6.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises an exogenous polynucleotidethat encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises a genetic modification in anendogenous polynucleotide that encodes a transcription factor involvedin glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the genetic modification is a truncation of the MTH1transcription factor. In an embodiment, the MTH1 transcription factormay have the amino acid sequence as set forth in SEQ ID NO: 1 and thetruncated MTH1 transcription factor may have the amino acid sequence setforth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the truncated MTH1 transcription factor has a longerhalf-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofacetyl-CoA to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetyl-CoA to acetoacetate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion of1,2-propanediol to 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of 1,2-propanediol to 1-propanolinclude: one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of 1,2-propanediol to propionaldehyde, and/orone or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of propionaldehyde to 1-propanol.

The present disclosure also provides a non-naturally occurringmicroorganism comprising: one or more exogenous polynucleotides encodingone or more enzymes in a pathway that produces cytosolic acetyl-CoA; oneor more polynucleotides coding for an acetoacetyl-CoA hydrolase; one ormore polynucleotides coding for enzymes in a pathway that catalyzes aconversion of dihydroxyacetone phosphate to 1,2-propanediol or1-propanol or one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of lactate to 1,2-propanediol or1-propanol, and one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol, whereinthe microorganism has reduced levels of pyruvate decarboxylase enzymaticactivity, and wherein the microorganism is capable of growing on a C6sugar as a sole carbon source and under anaerobic conditions.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has no detectable pyruvate decarboxylaseenzymatic activity.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or morepolynucleotides that code for a transcription factor of an enzyme thatdecarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in each of the one ormore polynucleotides that code for enzymes that decarboxylate pyruvateor a disruption in each of the polynucleotides that code for atranscription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more polynucleotides is adeletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate code for pdc1, pdc5, and/or pdc6. In someembodiments of each or any of the above or below mentioned embodiments,the one or more polynucleotides that code for a transcription factor ofone or more enzymes that decarboxylates pyruvate code for pdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises an exogenous polynucleotidethat encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises a genetic modification in anendogenous polynucleotide that encodes a transcription factor involvedin glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the genetic modification is a truncation of the MTH1transcription factor. In an embodiment, the MTH1 transcription factormay have the amino acid sequence as set forth in SEQ ID NO: 1 and thetruncated MTH1 transcription factor may have the amino acid sequence setforth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the truncated MTH1 transcription factor has a longerhalf-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of lactate to 1,2-propanediolinclude: one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of lactate to lactoyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactoyl-CoA to lactaldehyde and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1,2-propanodiol include: one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol and/or one ormore polynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of lactate to 1-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of lactate to lactoyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactoyl-CoA to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1-propanol include: one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetyl-CoA to acetoacetate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.

C6 sugar as a sole carbon source

The present disclosure also provides a non-naturally occurringmicroorganism comprising: a disruption of one or more enzymes thatdecarboxylate pyruvate; a genetic modification that permits growth ofthe microorganism on a C6 molecule as a sole carbon source; one or moreexogenous polynucleotides encoding one or more enzymes in a pathway thatproduces cytosolic acetyl-CoA, one or more polynucleotides coding for anacetoacetyl-CoA hydrolase, one or more polynucleotides coding forenzymes in a pathway that catalyzes a conversion of dihydroxyacetonephosphate to 1,2-propanediol or 1-propanol, and one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of acetyl-CoA to 2-propanol, and optionally one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of lactate to 1,2-propanediol or 1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has no detectable pyruvate decarboxylaseenzymatic activity.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or morepolynucleotides that code for a transcription factor of an enzyme thatdecarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in each of the one ormore polynucleotides that code for enzymes that decarboxylate pyruvateor a disruption in each of the polynucleotides that code for atranscription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more polynucleotides is adeletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate code for pdc1, pdc5, and/or pdc6. In someembodiments of each or any of the above or below mentioned embodiments,the one or more polynucleotides that code for a transcription factor ofone or more enzymes that decarboxylates pyruvate code for pdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises an exogenous polynucleotidethat encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism comprises a genetic modification in anendogenous polynucleotide that encodes a transcription factor involvedin glucose import.

In some embodiments of each or any of the above or below mentionedembodiments, the genetic modification is a truncation of the MTH1transcription factor. In an embodiment, the MTH1 transcription factormay have the amino acid sequence as set forth in SEQ ID NO: 1 and thetruncated MTH1 transcription factor may have the amino acid sequence setforth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the truncated MTH1 transcription factor has a longerhalf-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1,2-propanediol include: one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of dihydroxyacetone-phosphate to1-propanol include: one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of acetyl-CoA to 2-propanol include:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetyl-CoA to acetoacetate, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.

The present disclosure also provides methods for co-producing1,2-propanediol or 1-propanol and 2-propanol from a fermentable carbonsource under anaerobic conditions, the method comprising: a.) providinga fermentable carbon source in substantially anaerobic culture media;and b.) contacting the fermentable carbon source with any of thenon-naturally occurring microorganisms disclosed herein in afermentation media, wherein the microorganism co-produces1,2-propanediol or 1-propanol and 2-propanol from the fermentable carbonsource.

In some embodiments of each or any of the above or below mentionedembodiments, the fermentable carbon source is sugarcane juice, sugarcanemolasses, hydrolyzed starch, hydrolyzed lignocellulosic materials,glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, orglycerol in any form or mixture thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the fermentable carbon source is a monosaccharide,oligosaccharide, or polysaccharide.

The present disclosure also provides methods of making a non-naturallyoccurring microorganism that lacks pyruvate decarboxylase enzymaticactivity, that is capable of growth on a C6 molecule as a sole carbonsource, and that is capable of producing 1,2-propanediol or 1-propanoland 2-propanol from a fermentable carbon source under anaerobicconditions, the method comprising: introducing a disruption in one ormore polynucleotides in the microorganism that encode enzymes thatdecarboxylate pyruvate; introducing a genetic modification in themicroorganism that decreases import of glucose into the microorganism;introducing into the microorganism one or more exogenous polynucleotidesencoding one or more enzymes in a pathway that produces cytosolicacetyl-CoA; introducing into the microorganism one or morepolynucleotides coding for an acetoacetyl-CoA hydrolase oracetoacetyl-Coa transferase; introducing into the microorganism one ormore polynucleotides coding for enzymes in a pathway that catalyzes aconversion of dihydroxyacetone phosphate or pyruvate to 1,2-propanediolor 1-propanol, and introducing into the microorganism one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of acetyl-CoA to 2-propanol, and optionally introducing intothe microorganism one or more polynucleotides coding for enzymes in apathway that catalyzes a conversion of lactate to 1,2-propanediol or1-propanol.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,one preferred embodiment is shown in the following figure. It should beunderstood, however, that the disclosure is not limited to the precisearrangements, examples and instrumentalities shown.

FIG. 1 depicts an exemplary pathway for the co-production of 2-propanoland 1,2-propanediol, where 1,2-propanediol is produced via adihydroxyacetone-phosphate intermediate.

FIG. 2 depicts an exemplary pathway for the co-production of1,2-propanediol and 2-propanol, where 1,2-propanediol is produced via aglyceraldehyde 3-phosphate intermediate.

FIG. 3 depicts an exemplary pathway for the co-production of 2-propanoland 1-propanol, where 1-propanol is produced via adihydroxyacetone-phosphate intermediate.

FIG. 4 depicts an exemplary pathway for the co-production of 2-propanoland 1-propanol, where 1-propanol is produced via aglyceraldehyde-3-phosphate intermediate.

FIG. 5 depicts a schematic representation of the anaerobic flask systemused in Example 1.

FIG. 6A-C summarizes the results of Example 1 showing restored anaerobicgrowth of the BRKY-272 strain compared to the control (BRKY-397), carbonsource consumption and metabolite profile. FIG. 6A shows increasedgrowth of the BRKY-272 strain compared to the control (BRKY-397) in theanaerobic flask system shown in FIG. 5. FIGS. 6B and FIG. 6C show thekynetics of glucose consumption and metabolite production in strainsBRKY-397 (control) and BRKY-272, respectively.

FIG. 7 summarizes the results of Example 3 showing co-production of2-propanol and 1,2-propanediol in an ethanol-null strain BRKY-399.

DETAILED DESCRIPTION

The present disclosure generally relates to microorganisms (e.g.,non-naturally occurring microorganisms) that comprise a geneticallymodified pathway and uses of the microorganisms for the conversion of afermentable carbon source to 2-propanol and 1-propanol and/or1,2-propanediol. Such microorganisms may comprise one or morepolynucleotides coding for enzymes that catalyze a conversion of afermentable carbon source to 2-propanol, one or more polynucleotidescoding for enzymes that catalyze a conversion of a fermentable carbonsource to 1,2-propanediol, one or more polynucleotides coding forenzymes that catalyse a conversion of 1,2-propanediol to 1-propanol.

This disclosure provides, in part, the discovery of novel anaerobicenzymatic pathways including, for example, novel combinations ofenzymatic pathways, for the production of 2-propanol and 1-propanoland/or 1,2-propanediol from a carbon source (e.g., a fermentable carbonsource).

The present disclosure provides microorganisms (e.g., S. cerevisiae) forthe co-production of 2-propanol and 1-propanol and/or 1,2-propanediol.Microorganisms may be modified so that they may co-produce 2-propanoland 1-propanol and/or 1,2-propanediol. In an embodiment, a microorganismmay have its native ethanol production reduced or elimiated (i.e., shutoff). In an embodiment, to eliminate ethanol production in themicroorganism the activity of pyruvate decarboxylase (i.e., the enzymewhich decarboxylates pyruvate and in the process makes acetaldehyde andCO₂) may be disrupted including, for example, knocked-out. Pyruvatedecarboxylase comes in three isoforms in yeast and its activity can bemostly knocked out by deleting the genes PDC1, PDC5, and PDC6. Withoutwishing to be bound by a theory of the invention, the elimination of thepyruvate decarboxylase activity in the cell's cytoplasm renders theyeast cell unable to grow under anaerobic conditions due to two factors:(1) the lack of an alternative route for cytoplasmic acetyl-CoAproduction, due to the lack of acetaldehyde that would be converted toacetate and acetyl-coA; and (2) a redox imbalance due to excess NADHbecause the NADH is no longer oxidized in the conversion of acetaldehydeto ethanol. Thus, it is necessary to also alter the ability of themicroorgansim to import glucose by truncating a transcription factor ofthe glucose importer called MTH1. This truncation then restores theability of the ΔPDC1,5,6 mutant microorganism to survive on C6 sugars.In an embodiment, one or more polynucleotides coding for a bacterialpyruvate formate lyase or cytosolic pyruvate dehydrogenase complex maybe inserted into the microorganism to convert pyruvate into Acetyl CoAin the cytosol. In an embodiment, the microorganism may be modified tocomprise one or more polynucleotides that code for enzymes in a pathwayfor the coproduction of 2-propanol and 1-propanol and/or1,2-propanediol. In a further embodiment, the microorganism may bemodified to comprise an acetoacetylCoA hydrolase. Such an acetoacetylCoAhydrolase may be engineered from an acetoacetylCoA:acetate transferaseby making a single Glu-Asp mutation in the acetoacetylCoA:acetatetransferase (e.g., a E51 D Glu-Asp mutation corresponding to thenumbering of SEQ ID NO: 3). In an additional embodiment, a microorganismmay be modified to comprise one or more polynucleotides coding for aB12-independent dehydratase from the organism Roseburia inuvolurans toconvert 1,2-propanediol to propanaldehyde. Microorganims that compriseone or more of the modifications set forth above are termed anon-naturally occuring microroganism or a modified microorganism.

WO2004099425 discloses the overproduction of pyruvate in S. cerevisiaeby knocking out pyruvate decarboxylase activity and a directed evolutionprocess that allowed this triple mutant to grow on glucose due to atruncation of the MTH1 transcription factor. However, the scope stoppedat the overproduction of pyruvate in aerobic fermentation systems. Theuse of oxygen, in this context, was essential as there is a huge buildupof NADH in the cell due to the fact that NADH is no longer oxidized inthe conversion of acetaldehyde to ethanol.

The present disclosure further comprises a pyruvate overproducing cellable to produce cytosolic Acetyl-CoA inserting for example, bacterialpyruvate formate lyase or cytosolic pyruvate dehydrogenase complex toconvert pyruvate into Acetyl-CoA in the cytosol of the eukaryote cell.The insertion of pyruvate formate lyase in to a PDC-negative yeaststrain was disclosed by Waks and Silver in Engineering a SyntheticDual-Organism System for Hydrogen Production (Applied and EnvironmentalMicrobiology, vol. 75, n. 7, 2009, p. 1867-1875) without success inanaerobic growth or metabolism. Furthermore, the present disclosurefurther comprises a pyruvate overproducing cell able to producecytosolic Acetyl-CoA and to grow under anaerobic conditions by providinga temporary redox sink that allows reoxidation of NADH by introducing agene coding for a bacterial soluble NAD(P)+ transhydrogenase(Si-specific) (udhA gene from E. coli, E.C. number 1.6.1.1.) thatcatalyzes the interconversion of NADP++NADH=NADPH+NAD+. The concomitantexpression of the PFL and udhA enzymes to restore anaerobic growth tothe PDC-null yeast strain expressing the truncated MTH1 constitutes thefirst report of anaerobic growth of a PDC-null yeast strain and servesas a new eukaryotic chassis for the production of commodity chemicals.

Moreover, the present disclosure teaches how to make the 1,2-propanol or1-propanol and 2-propanol pathways work in the new eukaryote chassis.Since the cell had the production of acetaldehyde knocked out, acetateis no longer formed and a new CoA receptor is necessary for the2-propanol metabolic pathway to work. To solve this matter, the presentdisclosure proposes, for example, to engineer an acetoacetyl-CoAhydrolase from an acetoacetyl-CoA:acetate transferase (EC number2.8.3.8.) by applying a mutation to it that was reported by Mack andBuckel in Conversion of glutaconate CoA-transferase from Acidaminococcusfermentans into an acyl-CoA hydrolase by site-directed mutagenesis (FEBSLetters, v. 405, n. 2, 1997, p. 209-212) but applied to anothertransferase. In that case, the “glucatonate CoA transferase” wastransformed into a hydrolase by a single Glu-Asp mutation. The mainadvantage of this strategy is that the specificity of the enzyme foracetoacetyl-CoA is maintained since the transferase activity of aprotein that already has high specificity for acetoacetyl-CoA is knockedout. The methods provided herein may also provide end-results similar tothose of sterilization without the high capital expenditure andcontinuing higher management costs required to establish and maintainsterility throughout a production process. In this regard, mostindustrial-scale isoprene production processes are operated in thepresence of measurable numbers of bacterial contaminants. Such drawbacksof prior methods are avoided by the presently disclosed methods as thetoxic nature of the produced 2-propanol and/or 1-propanol reducecontaminants in the production process.

Additionally, the non-naturally occurring eukaryotic microorganismdisclosed herein is capable of anaerobic growth and concomitantproduction of 2-propanol and 1-propanol and/or 1,2-propanediol. Thesupplementation of oxygen and nitrogen in a fermenter requires anadditional investment for aerobic process. Additionally, aerobicfermentation processes for the production of 2-propanol and 1-propanoland/or 1,2-propanediol present several drawbacks at industrial scale(where it is technically challenging to maintain aseptic conditions)such as the fact that: (i) greater biomass is obtained reducing overallyields on carbon; (ii) the presence of oxygen favors the growth ofcontaminants (Weusthuis et al., 2011, Trends in Biotechnology, 2011,Vol. 29, No. 4, 153-158) and (iii) the mixture of oxygen and gaseouscompounds poses serious risks of explosion, (iv) the oxygen can catalyzethe unwanted reaction of polymerization of the olefinic compounds and,finally, (v) higher costs of fermentation and purification in aerobicconditions. Each of the drawbacks associated with aerobic fermentationincluding, for example, the risk of an explosion during the manufactureof 2-propanol and 1-propanol and/or 1,2-propanediol including dilutionby oxygen and nitrogen are overcome by the anaerobic fermentationmethods provided herein.

The present disclosure provides microorganisms comprising one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of the fermentable carbon source to one or more intermediatesin a pathway for the co-production of 2-propanol and 1-propanol and/or1,2-propanediol, and one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of the one or more intermediates to2-propanol and 1-propanol and/or 1,2-propanediol in a fermentationmedia, wherein 1,2-propanediol and 1-propanol are produced via adihydroxyacetone phosphate intermediate or a pyruvate intermediate. Insome embodiments, 2-propanol is produced via an acetyl-CoA intermediate.

The present disclosure also provides methods of co-producing 2-propanoland 1-propanol and/or 1,2-propanediol from a fermentable carbon sourceby providing a fermentable carbon source; contacting the fermentablecarbon source with a microorganism comprising one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of the fermentable carbon source to one or more intermediatesin a pathway for the co-production of 2-propanol and 1-propanol and/or1,2-propanediol, and one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of the one or more intermediates to2-propanol and 1-propanol and/or 1,2-propanediol in a fermentationmedia; and expressing the one or more polynucleotides coding for theenzymes in the pathway that catalyzes a conversion of the fermentablecarbon source to one or more intermediates in a pathway for theco-production of 2-propanol and 1-propanol and/or 1,2-propanediol andone or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of the one or more intermediates to 2-propanol and1-propanol and/or 1,2-propanediol in the microorganism to produce12-propanol and 1-propanol and/or 1,2-propanediol, wherein 2-propanoland 1-propanol and/or 1,2-propanediol are produced via adihydroxyacetone phosphate intermediate and/or a pyruvate intermediate,and wherein the co-production method is anaerobic.

It will be understood that the steps involved in any and all of themethods described herein may be performed in any order and are not to belimited or restricted to the order in which they are particularlyrecited. For example, the present disclosure provides methods ofco-producing 2-propanol and 1-propanol and/or 1,2-propanediol from afermentable carbon source, comprising: providing a fermentable carbonsource; contacting the fermentable carbon source with a microorganismcomprising one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of the fermentable carbon source to one ormore intermediates in a pathway for the co-production of 2-propanol and1-propanol and/or 1,2-propanediol, and one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion of the one ormore intermediates to 2-propanol and 1-propanol and/or 1,2-propanediolin a fermentation media; and expressing the one or more polynucleotidescoding for the enzymes in the pathway that catalyzes a conversion of thefermentable carbon source to one or more intermediates in a pathway forthe co-production of 2-propanol and 1-propanol and/or 1,2-propanedioland one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of the one or more intermediates to 2-propanol and1-propanol and/or 1,2-propanediol in the microorganism to produce2-propanol and 1-propanol and/or 1,2-propanediol. As such, expression ofthe one or more polynucleotides coding for the enzymes in the pathwaythat catalyzes a conversion of the fermentable carbon source to one ormore intermediates in a pathway for the co-production of 2-propanol and1-propanol and/or 1,2-propanediol and one or more polynucleotides codingfor enzymes in a pathway that catalyze a conversion of the one or moreintermediates to 2-propanol and 1-propanol and/or 1,2-propanediol in themicroorganism to produce 2-propanol and 1-propanol and/or1,2-propanediol may be preformed prior to or after contacting thefermentable carbon source with a microorganism comprising one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of the fermentable carbon source to one or more intermediatesin a pathway for the co-production of 2-propanol and 1-propanol and/or1,2-propanediol, and one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of the one or more intermediates to2-propanol and 1-propanol and/or 1,2-propanediol in a fermentationmedia.

Any of the intermediates produced in any of the enzymatic pathwaysdisclosed herein may be an intermediate in the classical sense of theword in that they may be enzymatically converted to another intermediateor an end product. Alternatively, the intermediates themselves may beconsidered an end product.

As used herein, the term “biological activity” or “functional activity,”when referring to a protein, polypeptide or peptide, may mean that theprotein, polypeptide or peptide exhibits a functionality or propertythat is useful as relating to some biological process, pathway orreaction. Biological or functional activity can refer to, for example,an ability to interact or associate with (e.g., bind to) anotherpolypeptide or molecule, or it can refer to an ability to catalyze orregulate the interaction of other proteins or molecules (e.g., enzymaticreactions).

As used herein, the term “culturing” may refer to growing a populationof cells, e.g., microbial cells, under suitable conditions for growth,in a liquid or on solid medium.

As used herein, the term “derived from” may encompass the termsoriginated from, obtained from, obtainable from, isolated from, andcreated from, and generally indicates that one specified material findsits origin in another specified material or has features that can bedescribed with reference to the another specified material.

As used herein, “exogenous polynucleotide” refers to anydeoxyribonucleic acid that originates outside of the microorganism.

As used herein, the term “an expression vector” may refer to a DNAconstruct containing a polynucleotide or nucleic acid sequence encodinga polypeptide or protein, such as a DNA coding sequence (e.g. genesequence) that is operably linked to one or more suitable controlsequence(s) capable of affecting expression of the coding sequence in ahost. Such control sequences include a promoter to affect transcription,an optional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites, and sequences whichcontrol termination of transcription and translation. The vector may bea plasmid, cosmid, phage particle, bacterial artificial chromosome, orsimply a potential genomic insert. Once transformed into a suitablehost, the vector may replicate and function independently of the hostgenome (e.g., independent vector or plasmid), or may, in some instances,integrate into the genome itself (e.g., integrated vector). The plasmidis the most commonly used form of expression vector. However, thedisclosure is intended to include such other forms of expression vectorsthat serve equivalent functions and which are, or become, known in theart.

As used herein, the term “expression” may refer to the process by whicha polypeptide is produced based on a nucleic acid sequence encoding thepolypeptides (e.g., a gene). The process includes both transcription andtranslation.

As used herein, the term “gene” may refer to a DNA segment that isinvolved in producing a polypeptide or protein (e.g., fusion protein)and includes regions preceding and following the coding regions as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “heterologous,” with reference to a nucleicacid, polynucleotide, protein or peptide, may refer to a nucleic acid,polynucleotide, protein or peptide that does not naturally occur in aspecified cell, e.g., a host cell. It is intended that the termencompass proteins that are encoded by naturally occurring genes,mutated genes, and/or synthetic genes. In contrast, the term homologous,with reference to a nucleic acid, polynucleotide, protein or peptide,refers to a nucleic acid, polynucleotide, protein or peptide that occursnaturally in the cell.

As used herein, the term a “host cell” may refer to a cell or cell line,including a cell such as a microorganism which a recombinant expressionvector may be transfected for expression of a polypeptide or protein(e.g., fusion protein). Host cells include progeny of a single hostcell, and the progeny may not necessarily be completely identical (inmorphology or in total genomic DNA complement) to the original parentcell due to natural, accidental, or deliberate mutation. A host cell mayinclude cells transfected or transformed in vivo with an expressionvector.

As used herein, the term “introduced,” in the context of inserting anucleic acid sequence or a polynucleotide sequence into a cell, mayinclude transfection, transformation, or transduction and refers to theincorporation of a nucleic acid sequence or polynucleotide sequence intoa eukaryotic or prokaryotic cell wherein the nucleic acid sequence orpolynucleotide sequence may be incorporated into the genome of the cell(e.g., chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon.Non-naturally occurring microbial organisms of the disclosure cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely. Those skilled in the art willunderstand that the genetic alterations, including metabolicmodifications exemplified herein, are described with reference to asuitable host organism such as E. coli and their corresponding metabolicreactions or a suitable source organism for desired genetic materialsuch as genes for a desired metabolic pathway. However, given thecomplete genome sequencing of a wide variety of organisms and the highlevel of skill in the area of genomics, those skilled in the art willreadily be able to apply the teachings and guidance provided herein toessentially all other organisms. For example, the E. coli metabolicalterations exemplified herein can readily be applied to other speciesby incorporating the same or analogous encoding nucleic acid fromspecies other than the referenced species. Such genetic alterationsinclude, for example, genetic alterations of species homologs, ingeneral, and in particular, orthologs, paralogs or nonorthologous genedisplacements.

As used herein, the term “operably linked” may refer to a juxtapositionor arrangement of specified elements that allows them to perform inconcert to bring about an effect. For example, a promoter may beoperably linked to a coding sequence if it controls the transcription ofthe coding sequence.

As used herein, “1,2-propanediol” is intended to mean propylene glycolwith general formula CH₃CH(OH)CH₂OH (CAS number—57-55-6).

As used herein, “1-propanol” is intended to mean n-propanol with ageneral formula CH₃CH₂CH₂OH (CAS number—71-23-8).

As used herein, “2-propanol” is intended to mean isopropyl alcohol witha general formula CH₃CH₃CHOH (CAS number—67-63-0).

As used herein, the term “a promoter” may refer to a regulatory sequencethat is involved in binding RNA polymerase to initiate transcription ofa gene. A promoter may be an inducible promoter or a constitutivepromoter. An inducible promoter is a promoter that is active underenvironmental or developmental regulatory conditions.

As used herein, the term “a polynucleotide” or “nucleic acid sequence”may refer to a polymeric form of nucleotides of any length and anythree-dimensional structure and single- or multi-stranded (e.g.,single-stranded, double-stranded, triple-helical, etc.), which containdeoxyribonucleotides, ribonucleotides, and/or analogs or modified formsof deoxyribonucleotides or ribonucleotides, including modifiednucleotides or bases or their analogs. Such polynucleiotides or nucleicacid sequences may encode amino acids (e.g., polypeptides or proteinssuch as fusion proteins). Because the genetic code is degenerate, morethan one codon may be used to encode a particular amino acid, and thepresent disclosure encompasses polynucleotides which encode a particularamino acid sequence. Any type of modified nucleotide or nucleotideanalog may be used, so long as the polynucleotide retains the desiredfunctionality under conditions of use, including modifications thatincrease nuclease resistance (e.g., deoxy, 2′-O-Me, phosphorothioates,etc.). Labels may also be incorporated for purposes of detection orcapture, for example, radioactive or nonradioactive labels or anchors,e.g., biotin. The term polynucleotide also includes peptide nucleicacids (PNA). Polynucleotides may be naturally occurring or non-naturallyoccurring. The terms polynucleotide, nucleic acid, and oligonucleotideare used herein interchangeably. Polynucleotides may contain RNA, DNA,or both, and/or modified forms and/or analogs thereof. A sequence ofnucleotides may be interrupted by non-nucleotide components. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replaced by P(O)S (thioate), P(S)S(dithioate), (O)NR₂ (amidate), P(O)R, P(O)OR′, COCH₂ (formacetal), inwhich each R or R′ is independently H or substituted or unsubstitutedalkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl,alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. Polynucleotides may be linear orcircular or comprise a combination of linear and circular portions.

As used herein, the term a “protein” or “polypeptide” may refer to acomposition comprised of amino acids and recognized as a protein bythose of skill in the art. The conventional one-letter or three-lettercode for amino acid residues is used herein. The terms protein andpolypeptide are used interchangeably herein to refer to polymers ofamino acids of any length, including those comprising linked (e.g.,fused) peptides/polypeptides (e.g., fusion proteins). The polymer may belinear or branched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art.

As used herein, related proteins, polypeptides or peptides may encompassvariant proteins, polypeptides or peptides. Variant proteins,polypeptides or peptides differ from a parent protein, polypeptide orpeptide and/or from one another by a small number of amino acidresidues. In some embodiments, the number of different amino acidresidues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or50. In some embodiments, variants differ by about 1 to about 10 aminoacids. Alternatively or additionally, variants may have a specifieddegree of sequence identity with a reference protein or nucleic acid,e.g., as determined using a sequence alignment tool, such as BLAST,ALIGN, and CLUSTAL (see, infra). For example, variant proteins ornucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with areference sequence.

As used herein, the term “recovered,” “isolated,” “purified,” and“separated” may refer to a material (e.g., a protein, peptide, nucleicacid, polynucleotide or cell) that is removed from at least onecomponent with which it is naturally associated. For example, theseterms may refer to a material which is substantially or essentially freefrom components which normally accompany it as found in its nativestate, such as, for example, an intact biological system.

As used herein, the term “recombinant” may refer to nucleic acidsequences or polynucleotides, polypeptides or proteins, and cells basedthereon, that have been manipulated by man such that they are not thesame as nucleic acids, polypeptides, and cells as found in nature.Recombinant may also refer to genetic material (e.g., nucleic acidsequences or polynucleotides, the polypeptides or proteins they encode,and vectors and cells comprising such nucleic acid sequences orpolynucleotides) that has been modified to alter its sequence orexpression characteristics, such as by mutating the coding sequence toproduce an altered polypeptide, fusing the coding sequence to that ofanother coding sequence or gene, placing a gene under the control of adifferent promoter, expressing a gene in a heterologous organism,expressing a gene at decreased or elevated levels, expressing a geneconditionally or constitutively in manners different from its naturalexpression profile, and the like.

As used herein, the term “selective marker” or “selectable marker” mayrefer to a gene capable of expression in a host cell that allows forease of selection of those hosts containing an introduced nucleic acidsequence, polynucleotide or vector. Examples of selectable markersinclude but are not limited to antimicrobial substances (e.g.,hygromycin, bleomycin, or chloramphenicol) and/or genes that confer ametabolic advantage, such as a nutritional advantage, on the host cell.

As used herein, the term “substantially anaerobic” means that growth ofthe modified micororganism takes place in culture media that comprises adissolved oxygen concentration of less than 5 ppm.

As used herein, the term “substantially similar” and “substantiallyidentical” in the context of at least two nucleic acids,polynucleotides, proteins or polypeptides may mean that a nucleic acid,polynucleotide, protein or polypeptide comprises a sequence that has atleast about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even99.5% sequence identity, in comparison with a reference (e.g.,wild-type) nucleic acid, polynucleotide, protein or polypeptide.Sequence identity may be determined using known programs such as BLAST,ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altshul et al.(1990) J. Mol. Biol. 215:403-410; Henikoff et al. (1989) Proc. Natl.Acad. Sci. 89:10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90:5873;and Higgins et al. (1988) Gene 73:237). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. Also, databases may be searched using FASTA(Person et al. (1988) Proc. Natl. Acad. Sci. 85:2444-2448.) In someembodiments, substantially identical polypeptides differ only by one ormore conservative amino acid substitutions. In some embodiments,substantially identical polypeptides are immunologically cross-reactive.In some embodiments, substantially identical nucleic acid moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, the term “transfection” or “transformation” may refer tothe insertion of an exogenous nucleic acid or polynucleotide into a hostcell. The exogenous nucleic acid or polynucleotide may be maintained asa non-integrated vector, for example, a plasmid, or alternatively, maybe integrated into the host cell genome. The term transfecting ortransfection is intended to encompass all conventional techniques forintroducing nucleic acid or polynucleotide into host cells. Examples oftransfection techniques include, but are not limited to, calciumphosphate precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, and microinjection.

As used herein, the term “transformed,” “stably transformed,” and“transgenic” may refer to a cell that has a non-native (e.g.,heterologous) nucleic acid sequence or polynucleotide sequenceintegrated into its genome or as an episomal plasmid that is maintainedthrough multiple generations.

As used herein, the term “vector” may refer to a polynucleotide sequencedesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmids,phage particles, single and double stranded cassettes and the like.

As used herein, the term “wild-type,” “native,” or “naturally-occurring”proteins may refer to those proteins found in nature. The termswild-type sequence refers to an amino acid or nucleic acid sequence thatis found in nature or naturally occurring. In some embodiments, awild-type sequence is the starting point of a protein engineeringproject, for example, production of variant proteins.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Singleton, et al.,Dictionary of Microbiology and Molecular Biology, second ed., John Wileyand Sons, New York (1994), and Hale & Markham, The Harper CollinsDictionary of Biology, Harper Perennial, NY (1991) provide one of skillwith a general dictionary of many of the terms used in this disclosure.Further, it will be understood that any of the substrates disclosed inany of the pathways herein may alternatively include the anion or thecation of the substrate.

Numeric ranges provided herein are inclusive of the numbers defining therange.

Unless otherwise indicated, nucleic acids sequences are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively.

While the present disclosure is capable of being embodied in variousforms, the description below of several embodiments is made with theunderstanding that the present disclosure is to be considered as anexemplification of the disclosure, and is not intended to limit thedisclosure to the specific embodiments illustrated. Headings areprovided for convenience only and are not to be construed to limit thedisclosure in any manner. Embodiments illustrated under any heading maybe combined with embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specifiedin this application, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” Also, thedisclosure of ranges is intended as a continuous range including everyvalue between the minimum and maximum values recited as well as anyranges that can be formed by such values. Also disclosed herein are anyand all ratios (and ranges of any such ratios) that can be formed bydividing a disclosed numeric value into any other disclosed numericvalue. Accordingly, the skilled person will appreciate that many suchratios, ranges, and ranges of ratios can be unambiguously derived fromthe numerical values presented herein and in all instances such ratios,ranges, and ranges of ratios represent various embodiments of thepresent disclosure.

Modification of Microorganism

A microorganism may be modified (e.g., genetically engineered) by anymethod known in the art to comprise and/or express one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of a fermentable carbon source to one or more intermediatesin a pathway for the co-production of 2-propanol and 1-propanol and/or1,2-propanediol. Such enzymes may include any of those enzymes as setforth in FIGS. 1-4. For example, the microorganism may be modified tocomprise one or more polynucleotides coding for enzymes that catalyze aconversion of dihydroxyacetone phosphate and/or pyruvate to1,2-propanodiol or 1-propanol. Modified microorganisms may be referredto herein as non-naturally occuring microorganisms.

In some embodiments, the non-naturally microorganism may comprise one ormore exogenous polynucleotides encoding one or more enzymes in pathwaysfor the co-production of 2-propanol and 1-propanol and/or1,2-propanediol from a fermentable carbon source under anaerobic ormicro-anaerobic conditions.

In some embodiments, the non-naturally microorganism may comprise one ormore polynucleotides coding for enzymes in a pathway that catalyzes aconversion of pyruvate to 2-propanol including, for example, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of pyruvate to acetyl-CoA, one or more polynucleotides codingfor enzymes in a pathway that catalyze a conversion of acetyl-CoA toacetoacetyl-CoA, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of acetoacetyl-CoA to acetoacetate,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol. Enzymes catalyzing any of theseconversions may include, for example, those enzymes listed in Table 1.

In some embodiments, the non-naturally occurring microorganism maycomprise one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of dihydroxyacetone-phosphate to1,2-propanediol including, for example: one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofdihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofmethylglyoxal to hydroxyacetone, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of hydroxyacetone to1,2-propanediol, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to lactaldehydeand/or one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of lactaldehyde to 1,2-propanediol. Enzymescatalyzing any of these conversions may include, for example, thoseenzymes listed in Table 2.

In some embodiments, the non-naturally occurring microorganism maycomprise one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of lactate to 1,2-propanediol including, forexample, one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of lactate to lactoyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactoyl-CoA to lactaldehyde and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactaldehyde to 1,2-propanediol. Enzymes catalyzing any ofthese conversions may include, for example, those enzymes listed inTable 3.

A modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to cytosolic acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of AcAcetate to acetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetone to 2-propanol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of dihydroxyacetone phosphate to methylglyoxal,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to hydroxyacetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of hydroxyacetone to 1,2-propanediol, and/or

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments, the modified microorganism has a disruption in theone or more polynucleotides that code for enzymes that decarboxylatepyruvate and associated transcription factor (e.g., pyruvatedecarboxylase 1, 2, 5, and 6). In some embodiments, the modifiedmicroorganism has a disruption in each polynucleotide that codes forenzymes that decarboxylate pyruvate and associated transcription factor(e.g., pyruvate decarboxylase 1, 2, 5, and 6). In some embodiments, themodified microorganism is capable of growth on a C6 sugar as a solecarbon source under anaerobic conditions. In some embodiments, themodified microorganism has a disruption in the one or morepolynucleotides that code for enzymes that decarboxylate pyruvate andassociated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5,and 6) and is capable of growth on a C6 sugar as a sole carbon sourceunder anaerobic conditions. In some embodiments, the modifiedmicroorganism has a disruption in each polynucleotide that codes forenzymes that decarboxylate pyruvate and associated transcription factor(e.g., pyruvate decarboxylase 1, 2, 5, and 6) and is capable of growthon a C6 sugar as a sole carbon source under anaerobic conditions.

A modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to lactate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactate to lactoyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactoyl-CoA to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of AcAcetate to acetone, and/or

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetone to 2-propanol.

In some embodiments, the modified microorganism has a disruption in eachof the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6). Insome embodiments, the modified microorganism is capable of growth on aC6 sugar as a sole carbon source under anaerobic conditions. In someembodiments, the modified microorganism has a disruption in each of theone or more polynucleotides that code for enzymes that decarboxylatepyruvate (e.g., pyruvate decarboxylase 1, 5, and 6) and is capable ofgrowth on a C6 sugar as a sole carbon source under anaerobic conditions.

A modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to cytosolic acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of AcAcetate to acetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetone to 2-propanol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of dihydroxyacetone phosphate to methylglyoxal,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to hydroxyacetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of hydroxyacetone to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to lactate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactate to lactoyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactoyl-CoA to lactaldehyde, and/or

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments, the modified microorganism has a disruption in eachof the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6). Insome embodiments, the modified microorganism is capable of growth on aC6 sugar as a sole carbon source under anaerobic conditions. In someembodiments, the modified microorganism has a disruption in each of theone or more polynucleotides that code for enzymes that decarboxylatepyruvate (e.g., pyruvate decarboxylase 1, 5, and 6) and is capable ofgrowth on a C6 sugar as a sole carbon source under anaerobic conditions.

In some embodiments, the non-naturally occurring microorganism maycomprise one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanolincluding, for example: one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol. Enzymes catalyzing any of these conversions may include, forexample, those enzymes listed in Table 2.

In some embodiments, the non-naturally occurring microorganism maycomprise one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of lactate to 1-propanol including, forexample, one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of lactate to lactoyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactoyl-CoA to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol. Enzymes catalyzing any of these conversions may include, forexample, those enzymes listed in Table 3.

A modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to cytosolic acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of AcAcetate to acetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetone to 2-propanol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of dihydroxyacetone phosphate to methylglyoxal,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to hydroxyacetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of hydroxyacetone to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of 1,2-propanediol to propionaldehyde, and/or

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of propionaldehyde to 1-propanol.

In some embodiments, the modified microorganism has a disruption in eachof the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate and associated transcription factor (e.g.,pyruvate decarboxylase 1, 2, 5, and 6). In some embodiments, themodified microorganism is capable of growth on a C6 sugar as a solecarbon source under anaerobic conditions. In some embodiments, themodified microorganism has a disruption in each of the one or morepolynucleotides that code for enzymes that decarboxylate pyruvate andassociated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5,and 6) and is capable of growth on a C6 sugar as a sole carbon sourceunder anaerobic conditions.

A modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to lactate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactate to lactoyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactoyl-CoA to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of 1,2-propanediol to propionaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of propionaldehyde to 1-propanol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of AcAcetate to acetone, and/or

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetone to 2-propanol.

In some embodiments, the modified microorganism has a disruption in eachof the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6). Insome embodiments, the modified microorganism is capable of growth on aC6 sugar as a sole carbon source under anaerobic conditions. In someembodiments, the modified microorganism has a disruption in each of theone or more polynucleotides that code for enzymes that decarboxylatepyruvate (e.g., pyruvate decarboxylase 1, 5, and 6) and is capable ofgrowth on a C6 sugar as a sole carbon source under anaerobic conditions.

modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to cytosolic acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of AcAcetate to acetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of acetone to 2-propanol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of dihydroxyacetone phosphate to methylglyoxal,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of methylglyoxal to hydroxyacetone,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of hydroxyacetone to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of 1,2-propanediol to propionaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of propionaldehyde to 1-propanol.

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of pyruvate to lactate,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactate to lactoyl-CoA,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactoyl-CoA to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of lactaldehyde to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of 1,2-propanediol to propionaldehyde, and/or

one or more polynucleotides coding for enzymes in a pathway thatcatalyzes a conversion of propionaldehyde to 1-propanol.

In some embodiments, the modified microorganism has a disruption in eachof the one or more polynucleotides that code for enzymes thatdecarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6). Insome embodiments, the modified microorganism is capable of growth on aC6 sugar as a sole carbon source under anaerobic conditions. In someembodiments, the modified microorganism has a disruption in each of theone or more polynucleotides that code for enzymes that decarboxylatepyruvate (e.g., pyruvate decarboxylase 1, 5, and 6) and is capable ofgrowth on a C6 sugar as a sole carbon source under anaerobic conditions.

Exemplary enzymes that convert a fermentable carbon source such asglucose to 1,2-propanediol (Pathway B1) and/or 2-propanol (Pathway A)and 1-propanol (Pathways B2 and C2) and/or 2-propanol (Pathway A)including, enzyme substrates, and enzyme reaction products associatedwith the conversions are presented in Tables 1 to 5 below. The enzymereference identifier listed in Tables 1 to 4 correlates with the enzymenumbering used in FIGS. 1-4, which schematically represents theenzymatic conversion of a fermentable carbon source such as glucose todihydroxyacetone phosphate or lactate and pyruvate. Dihydroxyacetonephosphate or lactate and pyruvate may be further converted to1,2-propanediol,1-propanol and/or 2-propanol, using any combination ofone or more enzymes provided in Tables 1 to 5 below including, all ofthose enzymes as provided in Tables 1 to 5 below.

TABLE 1 Pathway A (2-propanol from pyruvate) Enzyme EC No. Enzyme nameNumber Reaction A1. Formate-C 2.3.1.54 Pyruvate + CoA →acetyltransferase 1.97.1.4 Acetyl-CoA + formate Formate-Cacetyltransferase activating enzyme A2. Pyruvate dehydrogenase 1.2.4.1Pyruvate + CoA + NAD⁺ → 2.3.1.12 Acetyl-CoA + CO₂ + NADH 1.8.1.4 B.Thiolase 2.3.1.9 2 acetyl-CoA → acetoacetyl-CoA + CoA C. Acetoacetyl-CoA2.8.3.8 acetoacetyl-Coa + acetate → acetyltransferase acetoacetate +acetyl-CoA D. Acetatoacetate 4.1.1.4 acetoacetate → acetone + CO₂decarboxylase E. Secondary alcohol 1.1.1.2 acetone + NAD(P)H→dehydrogenase 2-propanol + NAD(P)+

TABLE 2 Pathway B1 (1,2-propanediol from Dihydroxyacetone phosphateEnzyme EC No. Enzyme name Number Reaction F1. methylglyoxal synthase4.2.3.3 dihydroxyacetone phosphate → methylglyoxal F2. methylglyoxalsynthase, 4.2.3.3 dihydroxyacetone phosphate → phosphate insensitivemethylglyoxal G. Methylglyoxal reductase 1.1.1.— Methylglyoxal →lactaldehyde H. Methylglyoxal reductase 1.1.1.78 methylglyoxal →hydroxyacetone I. methylglyoxal reductase 1.1.1.— Hydroxyacetone +NAD(P)H + H⁺ →1,2- [multifunctional] propanediol + NAD(P)⁺ J.methylglyoxal reductase 1.1.1.— Lactaldehyde + NAD(P)H + H⁺ →1,2-[multifunctional] propanediol + NAD(P)⁺

TABLE 3 Pathway C1 (1,2-propanediol from lactate) Enzyme EC No. Enzymename Number Reaction M1. D-Lactate dehydrogenase 1.1.1.28 Pyruvate +NAD(P)H + H⁺ −>D- Lactate + NAD(P)⁺ M2. L-Lactate dehydrogenase 1.1.1.27Pyruvate + NAD(P)H + H⁺ −>L- Lactate + NAD(P)⁺ N. propionate CoA-2.8.3.1 Lactate + Acetyl-CoA −> transferase* Lactoyl-CoA + Acetic acidO. Lactoyl-CoA synthase 2.3.3.— Lactate + CoA + ATP → lactoyl-CoA + AMPP. 1,2-propanediol 1.2.1.— Lactoyl-CoA + NAD(P)H + oxidoreductase H⁺ −>Lactaldehyde + NAD(P)⁺ Q. Lactaldehyde reductase 1.1.1.77L-Lactaldehyde + NAD(P)H + H⁺ −>L- 1,2-propanediol + NAD(P)⁺ J.methylglyoxal reductase 1.1.1.— Lactaldehyde + NAD(P)H + H⁺ −>1,2-[multifunctional] propanediol + NAD(P)⁺ *enzyme with homologous functionbut altered substrate specificity is required/preferred

TABLE 4 Pathway B2 (1-propanol from Dihydroxyacetone phosphate Enzyme ECNo. Enzyme name Number Reaction F1. methylglyoxal synthase 4.2.3.3dihydroxyacetone phosphate → methylglyoxal F2. methylglyoxal synthase,4.2.3.3 dihydroxyacetone phosphate → phosphate insensitive methylglyoxalG. Methylglyoxal reductase 1.1.1.— Methylglyoxal → lactaldehyde H.Methylglyoxal reductase 1.1.1.78 methylglyoxal → hydroxyacetone I.methylglyoxal reductase 1.1.1.— Hydroxyacetone + NAD(P)H + H⁺ →1,2-[multifunctional] propanediol + NAD(P)⁺ J. methylglyoxal reductase1.1.1.— Lactaldehyde + NAD(P)H + H⁺ →R/S [multifunctional]1,2-propanediol + NAD(P)⁺ K. 1,2 propanediol dehydratase 4.2.1.30 R/S1,2 propanediol → proprionaldehyde L. 1-propanol dehydrogenase 1.1.1.—proprionaldehyde + NADH → propanol + NAD+

TABLE 5 Pathway C2 (1-propanol from lactate) Enzyme EC No. Enzyme nameNumber Reaction M1. D-Lactate dehydrogenase 1.1.1.28 Pyruvate +NAD(P)H + H⁺ →D- Lactate + NAD(P)⁺ M2. L-Lactate dehydrogenase 1.1.1.27Pyruvate + NAD(P)H + H⁺ →L- Lactate + NAD(P)⁺ N. propionate CoA- 2.8.3.1Lactate + Acetyl-CoA → transferase* Lactoyl-CoA + Acetic acid O.Lactoyl-CoA synthase 2.3.3.— Lactate + CoA + ATP → lactoyl-CoA + AMP P.1,2-propanediol 1.2.1.— Lactoyl-CoA + NAD(P)H + oxidoreductase H⁺→Lactaldehyde + NAD(P)⁺ Q. Lactaldehyde reductase 1.1.1.77Lactaldehyde + NAD(P)H + H⁺ →1,2- propanediol + NAD(P)⁺ J. methylglyoxalreductase 1.1.1.— Lactaldehyde + NAD(P)H + H⁺ →R/S [multifunctional]1,2-propanediol + NAD(P)⁺ K. 1,2 propanediol 4.2.1.28 R/S 1,2propanediol → dehydratase proprionaldehyde L. 1-propanol dehydrogenase1.1.1.— proprionaldehyde + NADH → 1-propanol + NAD+ *enzyme withhomologous function but altered substrate specificity isrequired/preferred

The microorganism may be an archea, bacteria, or eukaryote. In someembodiments, the bacteria is a Propionibacterium, Propionispira,Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillusincluding, for example, Pelobacter propionicus, Clostridium propionicum,Clostridium acetobutylicum, Lactobacillus, Propionibacteriumacidipropionici or Propionibacterium freudenreichii. In someembodiments, the eukaryote is a yeast, filamentous fungi, protozoa, oralgae. In some embodiments, the yeast is Saccharomyces cerevisiae,Kluyveromyces lactis or Pichia pastoris.

In some embodiments, the microorganism is additionally modified tocomprise one or more tolerance mechanisms including, for example,tolerance to a produced molecule (i.e., methylglyoxal, 1-propanol, or2-propanol), and/or organic solvents. A microorganism modified tocomprise such a tolerance mechanism may provide a means to increasetiters of fermentations and/or may control contamination in anindustrial scale process.

In some embodiments, the disclosure contemplates the modification (e.g.,engineering) of one or more of the enzymes provided herein. Suchmodification may be performed to redesign the substrate specificity ofthe enzyme and/or to modify (e.g., reduce) its activity against otherssubstrates in order to increase its selectivity for a given substrate.Additionally or alternatively, one or more enzymes as provided hereinmay be engineered to alter (e.g., enhance including, for example,increase its catalytic activity or its substrate specificity) one ormore of its properties, including acceptance of different co-factorssuch as NADH instead of NADPH.

In some embodiments, sequence alignment and comparative modeling ofproteins may be used to alter one or more of the enzymes disclosedherein. Homology modeling or comparative modeling refers to building anatomic-resolution model of the desired protein from its primary aminoacid sequence and an experimental three-dimensional structure of asimilar protein. This model may allow for the enzyme substrate bindingsite to be defined, and the identification of specific amino acidpositions that may be replaced to other natural amino acid in order toredesign its substrate specificity.

Variants or sequences having substantial identity or homology with thepolynucleotides encoding enzymes as disclosed herein may be utilized inthe practice of the disclosure. Such sequences can be referred to asvariants or modified sequences. That is, a polynucleotide sequence maybe modified yet still retain the ability to encode a polypeptideexhibiting the desired activity. Such variants or modified sequences arethus equivalents in the sense that they retain their intended function.Generally, the variant or modified sequence may comprise at least about40%-60%, preferably about 60%-80%, more preferably about 80%-90%, andeven more preferably about 90%-95% sequence identity with the nativesequence.

One example of such a variant is described in SEQ ID NO: 3 wherein anE51D Glu-Asp mutation that renders the coded acetoacetyl-CoA transferaseinto a acetoacetyl-CoA hydrolase. Further modifications to SEQ ID NO: 3through rational and/or random approaches may be further performed toimprove hydrolase activity.

In some embodiments, a microorganism may be modified to expressincluding, for example, overexpress, one or more enzymes as providedherein. The microorganism may be modified by genetic engineeringtechniques (i.e., recombinant technology), classical microbiologicaltechniques, or a combination of such techniques and can also includenaturally occurring genetic variants to produce a genetically modifiedmicroorganism. Some of such techniques are generally disclosed, forexample, in Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press; and Selifonova et al. (2001)Appl. Environ. Microbiol. 67(8):3645).

A genetically modified microorganism may include a microorganism inwhich a polynucleotide has been inserted, deleted or modified (i.e.,mutated; e.g., by insertion, deletion, substitution, and/or inversion ofnucleotides), in such a manner that such modifications provide thedesired effect of expression (e.g., over-expression) of one or moreenzymes as provided herein within the microorganism. Geneticmodifications which result in an increase in gene expression or functioncan be referred to as amplification, overproduction, overexpression,activation, enhancement, addition, or up-regulation of a gene. Additionof cloned genes to increase gene expression can include maintaining thecloned gene(s) on replicating plasmids or integrating the cloned gene(s)into the genome of the production organism. Furthermore, increasing theexpression of desired cloned genes can include operatively linking thecloned gene(s) to native or heterologous transcriptional controlelements.

Where desired, the expression of one or more of the genes providedherein are under the control of a regulatory sequence that controlsdirectly or indirectly the expression of the gene in a time-dependentfashion during a fermentation reaction.

In some embodiments, a microorganism is transformed or transfected witha genetic vehicle such as, an expression vector comprising an exogenouspolynucleotide sequence coding for the enzymes provided herein.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host may typically, but not always, comprise a replicationsystem (i.e. vector) recognized by the host, including the intendedpolynucleotide fragment encoding the desired polypeptide, and maypreferably, but not necessarily, also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide-encoding segment. Expression systems (expression vectors)may include, for example, an origin of replication or autonomouslyreplicating sequence (ARS) and expression control sequences, a promoter,an enhancer and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, mRNA stabilizing sequences,nucleotide sequences homologous to host chromosomal DNA, and/or amultiple cloning site. Signal peptides may also be included whereappropriate, preferably from secreted polypeptides of the same orrelated species, which allow the protein to cross and/or lodge in cellmembranes or be secreted from the cell.

The vectors can be constructed using standard methods (see, e.g.,Sambrook et al., Molecular Biology: A Laboratory Manual, Cold SpringHarbor, N.Y. 1989; and Ausubel, et al., Current Protocols in MolecularBiology, Greene Publishing, Co. N.Y, 1995).

The manipulation of polynucleotides of the present disclosure includingpolynucleotides coding for one or more of the enzymes disclosed hereinis typically carried out in recombinant vectors. Numerous vectors arepublicly available, including bacterial plasmids, bacteriophage,artificial chromosomes, episomal vectors and gene expression vectors,which can all be employed. A vector of use according to the disclosuremay be selected to accommodate a protein coding sequence of a desiredsize. A suitable host cell is transformed with the vector after in vitrocloning manipulations. Host cells may be prokaryotic, such as any of anumber of bacterial strains, or may be eukaryotic, such as yeast orother fungal cells, insect or amphibian cells, or mammalian cellsincluding, for example, rodent, simian or human cells. Each vectorcontains various functional components, which generally include acloning site, an origin of replication and at least one selectablemarker gene. If given vector is an expression vector, it additionallypossesses one or more of the following: enhancer element, promoter,transcription termination and signal sequences, each positioned in thevicinity of the cloning site, such that they are operatively linked tothe gene encoding a polypeptide repertoire member according to thedisclosure.

Vectors, including cloning and expression vectors, may contain nucleicacid sequences that enable the vector to replicate in one or moreselected host cells. For example, the sequence may be one that enablesthe vector to replicate independently of the host chromosomal DNA andmay include origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. For example, the origin of replication from theplasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micronplasmid origin is suitable for yeast, and various viral origins (e.g. SV40, adenovirus) are useful for cloning vectors in mammalian cells.Generally, the origin of replication is not needed for mammalianexpression vectors unless these are used in mammalian cells able toreplicate high levels of DNA, such as COS cells.

A cloning or expression vector may contain a selection gene alsoreferred to as a selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will therefore not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin,methotrexate, hygromycin, thiostrepton, apramycin or tetracycline,complement auxotrophic deficiencies, or supply critical nutrients notavailable in the growth media.

The replication of vectors may be performed in E. coli (e.g., strain TB1or TG1, DH5α, DH10β, JM110). An E. coli-selectable marker, for example,the β-lactamase gene that confers resistance to the antibioticampicillin, may be of use. These selectable markers can be obtained fromE. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 orpUC19, or pUC119.

Expression vectors may contain a promoter that is recognized by the hostorganism. The promoter may be operably linked to a coding sequence ofinterest. Such a promoter may be inducible or constitutive.Polynucleotides are operably linked when the polynucleotides are in arelationship permitting them to function in their intended manner.

Promoters suitable for use with prokaryotic hosts may include, forexample, the a-lactamase and lactose promoter systems, alkalinephosphatase, the tryptophan (trp) promoter system, the erythromycinpromoter, apramycin promoter, hygromycin promoter, methylenomycinpromoter and hybrid promoters such as the tac promoter. Moreover, hostconstitutive or inducible promoters may be used. Promoters for use inbacterial systems will also generally contain a Shine-Dalgarno sequenceoperably linked to the coding sequence.

Viral promoters obtained from the genomes of viruses include promotersfrom polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2 or 5),herpes simplex virus (thymidine kinase promoter), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus (e.g., MoMLV,or RSV LTR), Hepatiti B virus, Myeloproliferative sarcoma virus promoter(MPSV), VISNA, and Simian Virus 40 (SV40). Heterologous mammalianpromoters include, e.g., the actin promoter, immunoglobulin promoter,heat-shock protein promoters.

The early and late promoters of the SV40 virus are conveniently obtainedas a restriction fragment that also contains the SV40 viral origin ofreplication (see, e.g., Fiers et al., Nature, 273:113 (1978); Mulliganand Berg, Science, 209:1422-1427 (1980); and Pavlakis et al., Proc.Natl. Acad. Sci. USA, 78:7398-7402 (1981)). The immediate early promoterof the human cytomegalovirus (CMV) is conveniently obtained as a HindIII E restriction fragment (see, e.g., Greenaway et al., Gene,18:355-360 (1982)). A broad host range promoter, such as the SV40 earlypromoter or the Rous sarcoma virus LTR, is suitable for use in thepresent expression vectors.

Generally, a strong promoter may be employed to provide for high leveltranscription and expression of the desired product. Among theeukaryotic promoters that have been identified as strong promoters forhigh-level expression are the SV40 early promoter, adenovirus major latepromoter, mouse metallothionein-I promoter, Rous sarcoma virus longterminal repeat, and human cytomegalovirus immediate early promoter (CMVor CMV IE). In an embodiment, the promoter is a SV40 or a CMV earlypromoter.

The promoters employed may be constitutive or regulatable, e.g.,inducible. Exemplary inducible promoters include jun, fos andmetallothionein and heat shock promoters. One or both promoters of thetranscription units can be an inducible promoter. In an embodiment, theGFP is expressed from a constitutive promoter while an induciblepromoter drives transcription of the gene coding for one or more enzymesas disclosed herein and/or the amplifiable selectable marker.

The transcriptional regulatory region in higher eukaryotes may comprisean enhancer sequence. Many enhancer sequences from mammalian genes areknown e.g., from globin, elastase, albumin, α-fetoprotein and insulingenes. A suitable enhancer is an enhancer from a eukaryotic cell virus.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the enhancer of the cytomegalovirus immediate earlypromoter (Boshart et al. Cell 41:521 (1985)), the polyoma enhancer onthe late side of the replication origin, and adenovirus enhancers (seealso, e.g., Yaniv, Nature, 297:17-18 (1982) on enhancing elements foractivation of eukaryotic promoters). The enhancer sequences may beintroduced into the vector at a position 5′ or 3′ to the gene ofinterest, but is preferably located at a site 5′ to the promoter.

Yeast and mammalian expression vectors may contain prokaryotic sequencesthat facilitate the propagation of the vector in bacteria. Therefore,the vector may have other components such as an origin of replication(e.g., a nucleic acid sequence that enables the vector to replicate inone or more selected host cells), antibiotic resistance genes forselection in bacteria, and/or an amber stop codon which can permittranslation to read through the codon. Additional eukaryotic selectablegene(s) may be incorporated. Generally, in cloning vectors the origin ofreplication is one that enables the vector to replicate independently ofthe host chromosomal DNA, and includes origins of replication orautonomously replicating sequences. Such sequences are well known, e.g.,the ColE1 origin of replication in bacteria. Various viral origins(e.g., SV40, polyoma, adenovirus, VSV or BPV) are useful for cloningvectors in mammalian cells. Generally, a eukaryotic replicon is notneeded for expression in mammalian cells unless extrachromosomal(episomal) replication is intended (e.g., the SV40 origin may typicallybe used only because it contains the early promoter).

To facilitate insertion and expression of different genes coding for theenzymes as disclosed herein from the constructs and expression vectors,the constructs may be designed with at least one cloning site forinsertion of any gene coding for any enzyme disclosed herein. Thecloning site may be a multiple cloning site, e.g., containing multiplerestriction sites.

The plasmids may be propagated in bacterial host cells to prepare DNAstocks for subcloning steps or for introduction into eukaryotic hostcells. Transfection of eukaryotic host cells can be any performed by anymethod well known in the art. Transfection methods include lipofection,electroporation, calcium phosphate co-precipitation, rubidium chlorideor polycation mediated transfection, protoplast fusion andmicroinjection. Preferably, the transfection is a stable transfection.The transfection method that provides optimal transfection frequency andexpression of the construct in the particular host cell line and type,is favored. Suitable methods can be determined by routine procedures.For stable transfectants, the constructs are integrated so as to bestably maintained within the host chromosome.

Vectors may be introduced to selected host cells by any of a number ofsuitable methods known to those skilled in the art. For example, vectorconstructs may be introduced to appropriate cells by any of a number oftransformation methods for plasmid vectors. For example, standardcalcium-chloride-mediated bacterial transformation is still commonlyused to introduce naked DNA to bacteria (see, e.g., Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), but electroporation andconjugation may also be used (see, e.g., Ausubel et al., 1988, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).

For the introduction of vector constructs to yeast or other fungalcells, chemical transformation methods may be used (e.g., Rose et al.,1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). Transformed cells may be isolated onselective media appropriate to the selectable marker used.Alternatively, or in addition, plates or filters lifted from plates maybe scanned for GFP fluorescence to identify transformed clones.

For the introduction of vectors comprising differentially expressedsequences to mammalian cells, the method used may depend upon the formof the vector. Plasmid vectors may be introduced by any of a number oftransfection methods, including, for example, lipid-mediatedtransfection (“lipofection”), DEAE-dextran-mediated transfection,electroporation or calcium phosphate precipitation (see, e.g., Ausubelet al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y.).

Lipofection reagents and methods suitable for transient transfection ofa wide variety of transformed and non-transformed or primary cells arewidely available, making lipofection an attractive method of introducingconstructs to eukaryotic, and particularly mammalian cells in culture.For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™(Stratagene) kits are available. Other companies offering reagents andmethods for lipofection include Bio-Rad Laboratories, CLONTECH, GlenResearch, InVitrogen, JBL Scientific, MBI Fermentas, PanVera, Promega,Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

The host cell may be capable of expressing the construct encoding thedesired protein, processing the protein and transporting a secretedprotein to the cell surface for secretion. Processing includes co- andpost-translational modification such as leader peptide cleavage, GPIattachment, glycosylation, ubiquitination, and disulfide bond formation.Immortalized host cell cultures amenable to transfection and in vitrocell culture and of the kind typically employed in genetic engineeringare preferred. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (CO 7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 derivatives adapted for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977); babyhamster kidney cells (BHK, ATCC CCL 10); DHFR-Chinese hamster ovarycells (ATCC CRL-9096); dp12.CHO cells, a derivative of CHO/DHFR-(EP307,247 published 15 Mar. 1989); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci., 383:44-68 (1982)); PEER human acutelymphoblastic cell line (Ravid et al. Int. J. Cancer 25:705-710 (1980));MRC 5 cells; FS4 cells; human hepatoma line (Hep G2), human HT1080cells, KB cells, JW-2 cells, Detroit 6 cells, NIH-3T3 cells, hybridomaand myeloma cells. Embryonic cells used for generating transgenicanimals are also suitable (e.g., zygotes and embryonic stem cells).

Suitable host cells for cloning or expressing polynucleotides (e.g.,DNA) in vectors may include, for example, prokaryote, yeast, or highereukaryote cells. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E.coli cloning host is E. coli 294 (ATCC 31,446), although other strainssuch as E. coli B, E. coli X1776 (ATCC 31,537), E. coli JM110 (ATCC47,013) and E. coli W3110 (ATCC 27,325) are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast may be suitable cloning or expression hosts for vectorscomprising polynucleotides coding for one or more enzymes. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

When the enzyme is glycosylated, suitable host cells for expression maybe derived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyxmori (silk moth) have been identified. A variety of viral strains fortransfection are publicly available, e.g., the L-1 variant of Autographacalifomica NPV and the Bm-5 strain of Bombyx mori NPV, and such virusesmay be used as the virus herein according to the present disclosure,particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hostcells.

Examples of useful mammalian host cells are Chinese hamster ovary cells,including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamsterovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney CV1 line transformed by SV40 (CO 7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, (Graham et al., J. Gen Virol. 36: 59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells(TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for production of one or more enzymes asdisclosed herein or with polynucleotides coding for one or more enzymesas disclosed herein and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Host cells containing desired nucleic acid sequences coding for thedisclosed enzymes may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al., Meth. Enz. 58: 44,(1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Polynucleotides and Encoded Enzymes

Any known polynucleotide (e.g., gene) that codes for an enzyme orvariant thereof that is capable of catalyzing an enzymatic conversionincluding, for example, an enzyme as set forth in any one of Tables 1-5or FIGS. 1-4, is contemplated for use by the present disclosure. Suchpolynucleotides may be modified (e.g., genetically engineered) tomodulate (e.g., increase or decrease) the substrate specificity of anencoded enzyme, or the polynucleotides may be modified to change thesubstrate specificity of the encoded enzyme (e.g., a polynucleotide thatcodes for an enzyme with specificity for a substrate may be modifiedsuch that the enzyme has specificity for an alternative substrate).Preferred microorganisms may comprise polynucleotides coding for one ormore of the enzymes as set forth in Tables 1-105 and FIGS. 1-4.

Enzymes for catalyzing the conversions set forth in pathways A, B1, B2,C1, and C2 of Tables 1-5 and FIGS. 1-4 are categorized in Table 6 below.Enzyme numbers presented in Table 6 that are followed by a numeral,e.g., A1 or A2, represent alternative enzymes that can catalyze aparticular conversion and may be generally referred to throughout thisdisclosure and figures by the first letter that precedes the numeral,e.g., A.

TABLE 6 Exemplary Gene Identifier (GI) numbers and/or Uniprot numbersPath- Enzyme EC Uniprot ID SEQ ID NO. SEQ ID NO. way Figs. No. numberEnzyme Candidate Gene (aa) (nucleotide) (protein) A 1, 2, A1 2.3.1.54Formate-C acetyltransferase PFLB P75793 12 13 3, 4 A 1, 2, A1 1.97.1.4Formate-C acetyltransferase PFLA C4ZXZ6 14 15 3, 4 (activating enzyme) A1, 2, A1 2.3.1.54 Formate-C acetyltransferase PFLB K9LI23 16 17 3, 4 A1, 2, A1 1.97.1.4 Formate-C acetyltransferase PFLA Q6RFH6 18 19 3, 4(activating enzyme) A 1, 2, A2 1.2.4.1 Pyruvate dehydrogenase pda1P16387 20 21 3, 4 complex A 1, 2, A2 1.2.4.1 Pyruvate dehydrogenase pdb1P32473 22 23 3, 4 complex A 1, 2, A2 2.3.1.12 Pyruvate dehydrogenaselat1 P12695 24 25 3, 4 complex A 1, 2, A2 1.8.1.4 Pyruvate dehydrogenaselpd1 P09624 26 27 3, 4 complex A 1, 2, A2 N/A Pyruvate dehydrogenasepdx1 P16451 28 29 3, 4 complex A 1, 2, A2 1.2.4.1 Pyruvate dehydrogenasepdhA F2MRX7 30 31 3, 4 complex (E1 aplha) A 1, 2, A2 1.2.4.1 Pyruvatedehydrogenase pdhB F2MRX8 32 33 3, 4 complex (E2 beta) A 1, 2, A22.3.1.12 Pyruvate dehydrogenase aceF F2MRX9 34 35 3, 4 complex (E2) A 1,2, A2 1.8.1.4 Pyruvate dehydrogenase lpd F2MRY0 36 37 3, 4 complex (E3)A 1, 2, B 2.3.1.9 acetyl coenzyme A thlA P45359 38 39 3, 4acetyltransferase A 1, 2, B 2.3.1.9 acetyl coenzyme A Erg10 P45359 40 413, 4 acetyltransferase A 1, 2, C1 2.8.3.8 Acetyl-CoA:acetoacetate-CoAatoA P76459 42 43 3, 4 transferase subunit A 1, 2, C1 2.8.3.8Acetyl-CoA:acetoacetate-CoA atoD P76458 44 45 3, 4 transferase subunit A1, 2, C2 3.1.2.— Acyl-CoAthioesterase 2 atoA* NA 3 N/A 3, 4 A 1, 2, D4.1.1.4 acetoacetate decarboxylase adc P23670 46 47 3, 4 A 1, 2, D4.1.1.4 acetoacetate decarboxylase adc A6M020 48 49 3, 4 A 1, 2, E1.1.1.2 secondary alcohol adh P25984 50 51 3, 4 dehydrogenase A N/ATemporary 1.6.1.1. Soluble pyridine nucleotide udhA P27306 104 105 redoxsink transhydrogenase A N/A Temporary 1.1.1.67 Mannitol-2dehydrogenasemdh Q83VI5 106 107 redox sink B1/B2 1, 3 F1 4.2.3.3 methylglyoxalsynthase mgsA P42980 52 53 B1/B2 1, 3 F1 4.2.3.3 methylglyoxal synthasemgsA P0A731 54 55 B1/B2 1, 3 F2 4.2.3.3 methylglyoxal synthase mgsA*P0A731 56 57 B1/B2 1, 3 G1, I1 1.1.1.6 glycerol dehydrogenase gldAP0A9S5 58 59 B1/B2 1, 3 G2 1.1.1.283 gre2 Q12068 60 61 B1/B2 1, 3 G31.1.1.21 aldose reductase gre3 P38715 62 63 B1/B2 1, 3 G4 1.1.1.76/butanediol dehydrogenase budC Q9ZNN8 64 65 1.1.1.304 B1/B2 1, 3 G51.1.1.4 butanediol dehydrogenase bdh1 P39714 66 67 B1/B2 1, 3 H1 1.1.1.—Alcohol dehydrogenase yqhD* Q46856 68 69 B1/B2 1, 3 H2 1.1.1.—methylglyoxal reductase ydjg P77256 70 71 B1/B2 1, 3 H3 1.1.1.—methylglyoxal reductase ypr1 C7GMG9 72 73 B1/B2 1, 3 I2 1.1.1.304methylglyoxal reductase, budC Q48436 74 75 multifunctional B1/B2 1, 2,J1 1.1.1.77 lactaldehyde reductase fucO P0A9S1 76 77 3, 4 B1/B2 1, 2, J21.1.1.— methylglyoxal reductase yafB P30863 78 79 3, 4 [multifunctional]B2, C2 3, 4 K1 4.2.1.30 glycerol dehydratase dhaB1 Q8GEZ8 80 81 B2, C23, 4 K1 4.2.1.30 glycerol dehydratase activator dhaB2 Q8GEZ7 82 83 B2,C2 3, 4 K2 4.2.1.30 diol dehydratase b1 Q1A666 84 85 B2, C2 3, 4 K24.2.1.30 diol dehydratase activator b2 Q1A665 86 87 B2, C2 3, 4 L1.1.1.1 alcohol dehydrogenase adh C6PZV5 88 89 C1/C2 2, 4 M1 1.1.1.28D-Lactate dehydrogenase ldhA P52643 90 91 C1/C2 2, 4 M2 1.1.1.27L-Lactate dehydrogenase ldhL2 P59390 92 93 C1/C2 2, 4 M2 1.1.1.27L-lactate dehydrogenase ldh2 P19858 94 95 C1/C2 2, 4 N 2.8.3.1propionate CoA-transferase* pct Q9L3F7 96 97 C1/C2 2, 4 O 2.3.3.—Lactoyl-CoA Synthase ACS1 Q01574 98 99 C1/C2 2, 4 P 1.2.1.—CoA-dependent pduP Q9XDN1 100 101 propionaldehyde dehydrogenase* C1/C22, 4 Q 1.1.1.77 L-1,2-propanediol fucO P0A9S1 102 103 oxidoreductaseMethods for the Co-Production of 2-Propanol and 1-Propanol and/or1,2-Propanediol

2-propanol and 1-propanol and/or 1,2-propanediol may be produced bycontacting any of the genetically modified microorganisms providedherein with a fermentable carbon source. Such methods may preferablycomprise contacting a fermentable carbon source with a microorganismcomprising one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of the fermentable carbon source to any ofthe intermediates provided in FIGS. 1-4 (Tables 1-6) and one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of the one or more intermediates provided in FIGS. 1-4(tables 1-6) to 2-propanol and 1-propanol and/or 1,2-propanediolin afermentation media; and expressing the one or more polynucleotidescoding for the enzymes in the pathway that catalyzes a conversion of thefermentable carbon source to the one or more intermediates provided inFIGS. 1-4 (tables 1-6) and one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of the one or moreintermediates provided in FIGS. 1-4 (tables 1-6) to 2-propanol and1-propanol and/or 1,2-propanediol.

The metabolic pathways that lead to the production of industriallyimportant compounds involve oxidation-reduction (redox) reactions. Forexample, during fermentation, glucose is oxidized in a series ofenzymatic reactions into smaller molecules with the concomitant releaseof energy. The electrons released are transferred from one reaction toanother through universal electron carriers, such Nicotinamide AdenineDinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate(NAD(P)), which act as cofactors for oxidoreductase enzymes. Inmicrobial catabolism, glucose is oxidized by enzymes using the oxidizedform of the cofactors (NAD(P)+ and/or NAD+) thus generating reducingequivalents in the form of the reduced cofactor (NAD(P)H and NADH). Inorder for fermentation to continue, redox-balanced metabolism isrequired, i.e., the cofactors must be regenerated by the reduction ofmicrobial cell metabolic compounds.

Microorganism-catalyzed fermentation for the production of naturalproducts is a widely known application of biocatalysis. Industrialmicroorganisms can affect multistep conversions of renewable feedstocksto high value chemical products in a single reactor. Products ofmicroorganism-catalyzed fermentation processes range from chemicals suchas ethanol, lactic acid, amino acids and vitamins, to high value smallmolecule pharmaceuticals, protein pharmaceuticals, and industrialenzymes. In many of these processes, the biocatalysts are whole-cellmicroorganisms, including microorganisms that have been geneticallymodified to express heterologous genes.

Some key parameters for efficient microorganism-catalyzed fermentationprocesses include the ability to grow microorganisms to a greater celldensity, increased yield of desired products, increased amount ofvolumetric productivity, removal of unwanted co-metabolites, improvedutilization of inexpensive carbon and nitrogen sources, adaptation tovarying fermenter conditions, increased production of a primarymetabolite, increased production of a secondary metabolite, increasedtolerance to acidic conditions, increased tolerance to basic conditions,increased tolerance to organic solvents, increased tolerance to highsalt conditions and increased tolerance to high or low temperatures.Inefficiencies in any of these parameters can result in highmanufacturing costs, inability to capture or maintain market share,and/or failure to bring fermented end-products to market.

The methods and compositions of the present disclosure can be adapted toconventional fermentation bioreactors (e.g., batch, fed-batch, cellrecycle, and continuous fermentation).

In some embodiments, a microorganism (e.g., a genetically modifiedmicroorganism) as provided herein is cultivated in liquid fermentationmedia (i.e., a submerged culture) which leads to excretion of thefermented product(s) into the fermentation media. In one embodiment, thefermented end product(s) can be isolated from the fermentation mediausing any suitable method known in the art.

In some embodiments, formation of the fermented product occurs during aninitial, fast growth period of the microorganism. In one embodiment,formation of the fermented product occurs during a second period inwhich the culture is maintained in a slow-growing or non-growing state.In one embodiment, formation of the fermented product occurs during morethan one growth period of the microorganism. In such embodiments, theamount of fermented product formed per unit of time is generally afunction of the metabolic activity of the microorganism, thephysiological culture conditions (e.g., pH, temperature, mediumcomposition), and the amount of microorganisms present in thefermentation process.

In some embodiments, the fermentation product is recovered from theperiplasm or culture medium as a secreted metabolite. In one embodiment,the fermentation product is extracted from the microorganism, forexample when the microorganism lacks a secretory signal corresponding tothe fermentation product. In one embodiment, the microorganisms areruptured and the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions maythen be separated if necessary. The fermentation product of interest maythen be purified from the remaining supernatant solution or suspensionby, for example, distillation, fractionation, chromatography,precipitation, filtration, and the like.

The methods of the present disclosure are preferably preformed underanaerobic conditions. Both the degree of reduction of a product as wellas the ATP requirement of its synthesis determines whether a productionprocess is able to proceed aerobically or anaerobically. To produce2-propanol and 1-propanol and/or 1,2-propanediol via anaerobic microbialconversion, or at least by using a process with reduced oxygenconsumption, redox imbalances should be avoided. Several types ofmetabolic conversion steps involve redox reactions including some of theconversions as set forth in FIG. 1. Such redox reactions involveelectron transfer mediated by the participation of redox cofactors suchas NADH, NADPH and ferredoxin. Since the amounts of redox cofactors inthe cell are limited to permit the continuation of metabolic processes,the cofactors have to be regenerated. In order to avoid such redoximbalances, alternative ways of cofactor regeneration may be engineered,and in some cases additional sources of ATP generation may be provided.Alternatively, oxidation and reduction processes may be separatedspatially in bioelectrochemical systems (Rabaey and. Rozendal, 2010,Nature reviews, Microbiology, vol 8: 706-716).

In some embodiments, redox imbalances may be avoided by using substrates(e.g., fermentable carbon sources) that are more oxidized or morereduced. for example, if the utilization of a substrate results in adeficit or surplus of electrons, a requirement for oxygen can becircumvented by using substrates that are more reduced or oxidized,respectively. For example, glycerol which is a major byproduct ofbiodiesel production is more reduced than sugars, and is therefore moresuitable for the synthesis of compounds whose production from sugarresults in cofactor oxidation, such as succinic acid. In someembodiments, if the conversion of a substrate to a product results in anelectron deficit, co-substrates can be added that function as electrondonors (Babel 2009, Eng. Life Sci. 9,285-290). An important criterionfor the anaerobic use of co-substrates is that their redox potential ishigher than that of NADH (Geertman et al., 2006, FEMS Yeast Res. 6,1193-1203). If the conversion of substrate to produce results in anelectron surplus, co-substrates can be added that function as electronacceptors.

In some embodiments, a gene coding for an enzyme that can act as atemporary redox sink (i.e. by catalyzing a reduction reaction of areadily available substrate) is used in order to avoid redox imbalances.Some examples of genes that may act as redox sinks in yeast weredescribed in Wang et al., 2012, Biochem. Eng. J. vol. 67, p 126-131.These enzymes include, but are not limited to, soluble pyridinetranshydrogenases (EC number 1.6.1.1.) and water-forming NADH oxidase(EC number 1.6.3.4.).

Methods for the Production of Polypropylene

1-propanol and 2-propanol produced via methods disclosed herein andwithout the need of separating one from the other may be dehydratedtogether to form propylene, which may then be polymerized to producepolypropylene in a cost-effective manner.

Propylene is a chemical compound that is widely used to synthesize awide range of petrochemical products. For instance, this olefin is theraw material used for the production of polypropylene, its copolymersand other chemicals such as acrylonitrile, acrylic acid, epichloridrineand acetone. Propylene demand is growing faster than ethylene demand,mainly due to the growth of market demand for polypropylene. Propyleneis polymerized to produce thermoplastics resins for innumerousapplications such as rigid or flexible packaging materials, blow moldingand injection molding.

Propylene is typically obtained in large quantity scales as a byproductof catalytical or thermal oil cracking, or as a co-product of ethyleneproduction from natural gas. (Propylene, Jamie G. Lacson, CEH MarketingResearch Report-2004, Chemical Economics Handbook-SRI International).The use of alternative routes for the production of propylene has beencontinuously evaluated using a wide range of renewable raw materials(“Green Propylene”, Nexant, January 2009). These routes include, forexample, dimerization of ethylene to yield butylene, followed bymetathesis with additional ethylene to produce propylene. Another routeis biobutanol production by sugar fermentation followed by dehydrationand methatesis with ethylene. Some thermal routes are also beingevaluated such as gasification of biomass to produce a syngas followedby synthesis of methanol, which may then produce green propylene viamethanol-to-olefin technology.

Propylene production by 2-propanol dehydration has been well-describedin document EP00498573B1, wherein all examples show propyleneselectivity higher than 90% with high conversions. Dehydration of1-propanol has also been studied in the following articles: “Mechanismand Kinetics of the Acid-Catalyzed Dehydration of 1- and iso-propanol inHot Compressed Liquid Water” (Antal, M et al., Ind. Eng. Chem. Res.1998, 37, 3820-3829) and “Fischer-Tropsch Aqueous Phase Refining byCatalytic Alcohol Dehydration” (Nel, R. et al., Ind. Eng. Chem. Res.2007, 46, 3558-3565). The reported yield is higher than 90%.

Without further description, it is believed that one of ordinary skillin the art may, using the preceding description and the followingillustrative examples, make and utilize the agents of the presentdisclosure and practice the claimed methods. The following workingexamples are provided to facilitate the practice of the presentdisclosure, and are not to be construed as limiting in any way theremainder of the disclosure.

EXAMPLES Example 1 Modification of a Microorganism to Render it Unableto Produce Ethanol, but Able to Grow on Glucose as the Sole CarbonSource Under Anaerobic Conditions

This example demonstrates the construction of yeast strain BRKY-272(haploid and isogenic to Saccharomyces cerevisiae S288C) simultaneouslyexpressing genes coding for enzymes in a pathway that catalyze theconversion of an acetyl-CoA intermediate to 2-propanol and genes codingfor enzymes in a pathway that catalyzes the production of cytosolicacetyl-CoA intermediate from a pyruvate intermediate. The strain furthercomprises deletions of the PDC1, PDC5 and PDC6 genes coding for thethree pyruvate decarboxylase isoforms, and thus lacks pyruvatedecarboxylase activity and the capacity to produce ethanol. The strainfurther comprises an integration of a gene expressing the truncatedversion of the MTH1 enzyme as set forth in SEQ ID NO: 2.

The strains listed in Table 7 represent the step-wise creation of strainBRKY-272. All DNA-mediated transformation into S. cerevisiae wasconducted using the Lithium Acetate procedure as described by Gietz R Wand Woods R A, Guide to Yeast Gentics and Molecular Cell Biology. PartB. San Diego, Calif.: Academic Press Inc. pp. 87-96 (2002) and in allcases integration of the constructs was confirmed by PCR amplificationans sequencing of genomic DNA. In some cases, strains with more than onedesired trait were obtained by crossing haploid strains of compatiblemating types. In these cases, diploid construction, sporulation, tetraddissection, and random spore analysis was performed according to Treco DA and Winston F, UNIT 13.2 Growth and Manipulation of Yeast, Curr.Protoc. Mol. Biol. 82:13.2.1-13.2.12 (2008).

Strains representing the step-wise creation of strain BRKY-272 thatsimultaneously expresses all genes coding for enzymes in a pathway thatcatalyze the conversion of an acetyl-CoA intermediate to 2-propanol andgenes coding for enzymes in a pathway that catalyzes the production ofcytosolic acetyl-CoA intermediate from a pyruvate intermediate areprovided in Table 7 below. The strain further comprises deletions of thePDC1, PDC5 and PDC6 genes coding for the three pyruvate decarboxylaseisoforms and the integration of a gene coding for the truncated versionof the MTH1 gene.

TABLE 7 Strains for the step-wise creation of strain BRKY-272. Strain IDStrain Genotype Vectors Host FY23 S288C, mating type a, ura3-52, leu2Δ1,trp1Δ63 none N/A FY86 S288C, mating type alpha, ura3-52, leu2Δ1, noneN/A his3delta200 BRKY-02 S288C, mating type a, ura3-52, leu2Δ1, noneFY23 trp1Δ63, PDC1::URA3 BRKY-31 S288C, mating type a; his3delta200,ura3-52, none FY23 × FY86 trp1delta63, leu2delta1 progeny BRKY-37 S288C,mating type alpha; his3delta200, ura3-52, none FY86 × BRKY02trp1delta63, leu2delta1, PDC1::URA3 progeny BRKY-69 S288C, mating typea; his3delta200, ura3-52, none BRKY31 trp1delta63, leu2delta1,PDC6::URA3 BRKY-86 S288C, mating type a; his3delta200, ura3-52, noneBRKY69 trp1delta63, leu2delta1, PDC6::URA3, locus Chr XI: 91575-adh/HIS3BRKY-97 S288C, mating type a; his3delta200, ura3-52, none BRKY31trp1delta63, leu2delta1, PDC5::KanMX4_tMTH1 BRKY-115 S288c, mating typeα; his3delta200, ura3-52, none BRKY37 × BRKY97 trp1delta63, leu2delta1,PDC5::KanMX4_tMTH1 progeny BRKY-118 S288c, mating type alpha;his3delta200, ura3-52, none BRKY37 × BRKY97 trp1delta63, leu2delta1,progeny PDC5::KanMX4_tMTH1 PDC1::URA3 BRKY-130 S288C, mating type α;his3delta200, ura3-52, none BRKY115 × BRKY86 trp1delta63, leu2delta1,PDC5::KanMx4/MTH1, progeny PDC6::URA3, locus Chr XI: 91575-adh/HIS3BRKY-138 S288C, mating type a; his3delta200, ura3-52, none BRKY86 ×BRKY118 trp1delta63, leu2delta1, PDC1::URA3, progeny PDC5::KanMX4/MTH1,locus Chr XI: 91575- adh/HIS3 BRKY-163 S288C, mating type alpha;his3delta200, ura3-52, none BRKY138 × BRKY130 trp1delta63, leu2delta1,PDC1::URA3, progeny PDC5::KanMX4/MTH1, PDC6::URA3 locus Chr XI:91575-adh/HIS3 BRKY-174 S288C, mating type alpha; his3delta200, ura3-52,none BRKY163 trp1delta63, leu2delta1, PDC1::URA3, PDC5::KanMX4/MTH1,PDC6::URA3, locus Chr XI: 91575-adh/HIS3, locus ChrX: 194944-atoA/atoD/TRP1 BRKY-189 S288C, mating type alpha; his3delta200, ura3-52,none BRKY174 trp1delta63, leu2delta1, PDC1::URA3, PDC5::KanMX4/MTH1,PDC6::URA3, locus Chr XI: 91575-adh/HIS3, ChrX: 194944- atoA/atoD/TRP1,locus YPRCtau3::thl/adc/natMX BRKY 272 S288C, mating type α;his3delta200, ura3-52, pRS415-LEU2 with BRKY189 trp1delta63, leu2delta1,PDC1::URA3, genes pPGK1-PFLA PDC5::KanMX4/MTH1, PDC6::URA3, locus Chr(E. coli)-tADH1, XI: 91575-adh/HIS3 isolate 1.1, ChrX: 194944- pTEF-PFLB(E. coli)- atoA/atoD/TRP1 isolate 10, locus tTDH3, pTDH3-YPRCtau3::thl/adc/natMX isolate 20.1; LEU2, udhA-tADH1 pPGK1-PFLA (E.coli)-tADH1, pTEF-PFLB (E. coli)-tTDH3, pTDH3-udhA-tADH1 BRKY 397 S288C,mating type α; his3delta200, ura3-52, pRS415-LEU BRKY189 trp1delta63,leu2delta1, pdc1::URA3, (empty) pdc5::KanMX4/MTH1, pdc6::URA3, locus ChrXI: 91575-adh/HIS3, ChrX: 194944- atoA/atoD/TRP1, locusYPRCtau3::thl/adc/natMX; LEU2

FY23 and FY86 are haploid strains isogenic to Saccharomyces cerevisiaeS288C as described by Winston et al. 1995, Yeast, 11, issue 1, 53-55),each containing three auxotrophic markers, and were used as the“wild-type” strains for this study.

Strain BRKY-02 was obtained by deleting the PDC1 gene from strain FY23with a URA3 marker in a linear construct. The linear construct was builtby PCR amplification of the URA3 marker gene from the commercial vectorpESC-URA with primers BK0592 and BK0593 (Table 8) containing 40 bp 5′extensions corresponding to regions upstream and downstream of the PDC1locus (SEQ ID NO: 4). Upon introduction in a S. cerevisiae host cell,this construct can integrated by homologous recombination into the PDC1locus of the genome, functionally disrupting PDC1p by replacing thePDC1p coding sequence with its integrating sequence. The resultingstrain was selected for uracyl prototrophy in Yeast Nitrogen Base Mediawithout uracyl (Sigma) and confirmed by PCR amplification of genomicDNA.

Strains BRKY-31 and BRKY-37 were generated by crossing and tetraddissection of strains FY23xFY86 and FY86xBRKY-02, respectively. Theobjective was to obtain haploid strains with four auxotrophic markersand, in the case of BRKY-37, the four auxotrophic markers and thePDC1::URA3 deletion.

Strain BRKY-69 was obtained by deleting the PDC6 gene from strainBRKY-31 with a URA3 marker in a linear construct. The linear constructwas built by PCR amplification of the URA3 marker gene from thecommercial vector pESC-URA with primers BK0678 and BK0679 (Table 8)containing 40 bp 5′ extensions corresponding to regions upstream anddownstream of the PDC6 locus (SEQ ID NO:5). Upon introduction into a S.cerevisiae host cell, this construct can integrate by homologousrecombination into the PDC6 locus of the genome, functionally disruptingPDC6p by replacing the PDC6p coding sequence with its integratingsequence. The resulting strain was selected for uracyl prototrophy inYeast Nitrogen Base Media without uracyl (Sigma) and confirmed by PCRamplification of genomic DNA.

Strain BRKY-86 was obtained by integrating at locus Chr XI:91575-..92913of strain BRKY-69 a construct for the expression of the secondaryalcohol dehydrogenase from Clostridium beijerinckii (Table 6, Enzyme No.E, E.C. Number 1.1.1.2) controlled by the TEF1 promoter and the PGK1terminator. The integration cassette was flanked by ˜150 bp homologyregions for locus Chr XI: 91575..92913 and comprised the HIS3auxotrophic marker for strain selection. The whole construct was builtby overlapping PCR (SEQ ID NO:6). Upon introduction in a S. cerevisiaehost cell, this construct can integrate by homologous recombination intothe Chr XI:91575-..92913 locus of the genome. The resulting strain wasselected for histidine prototrophy in Yeast Nitrogen Base Media withouthistidine (Sigma) and confirmed by PCR amplification and sequencing ofgenomic DNA.

Strain BKRY-97 was obtained by deleting the PDC5 gene from strainBRKY-31 with a linear construct comprising the KanMX4 marker gene and aexpression cassette for the truncated version of the MTH1 enzyme setforth in SEQ ID NO:2 flanked by homology sequences consisting of theupstream and downstream nucleotide sequences of the S. cerevisiae PDC5locus. A truncated version of the MTH1 gene was synthesized by IDT(Integrated DNA Technologies, Coralville, Iowa). Next, the truncatedMTH1 gene was introduced into a surrogate plasmid vector carrying theKanMX4 selection marker flanked by loxp recognition sequences (pUG6vector by Euroscarf). PCR amplification of the truncated MTH1 gene andKanMX4 marker gene from the surrogate vector was performed with primersBKO909 and BKO910 (Table 8) containing 5′ extensions corresponding toregions upstream and downstream of the PDC5 locus (SEQ ID NO:7). Uponintroduction in a S. cerevisiae host cell, this construct can integrateby homologous recombination into the PDC5 locus of the genome,functionally disrupting PDC5p by replacing the PDC5p coding sequencewith its integrating sequence for the concomitant expression of thetruncated version of MTH1 and KanMX4. The resulting strain was selectedfor Geneticin resistance in YPD Media containing 150 micrograms perliter of Geneticin and confirmed by PCR amplification and sequencing ofgenomic DNA.

Strain BRKY-115 is a progeny of a cross between strains BRKY-37 andBRKY-97 and differs from its parent BRKY-97 only in the mating type.This different mating type was used in a further cross aimed atobtaining the PDC-null triple deletion strain.

Strain BRKY-118 is an ethanol-null strain carrying deletions in genesPDC1 and PDC5, coding for the two most important isoforms of pyruvatedecarboxylases. Deletion of both genes causes significantly decreasedamounts of ethanol from sugar and creates a C2-auxotrophy. Aftersequential deletion of the two genes failed repeatedly, we performed across between strains BRKY-37 and BRKY-97 and selected tetrads on thebasis of uracyl prototrophy and Geneticin resistance and were able toobtain the PDC1/PDC5 double knockout.

Strain BRKY-130 is the progeny of a cross between BRKY115×BRKY86 andcarries deletions of PDC6 and PDC5 with the concomitant expression ofthe truncated version of the MTH1 and the secondary alcoholdehydrogenase of C. beijerinckii of the 2-propanol pathway.

Strain BRKY-138 is the progeny of a cross between BRKY86 and BRKY118 andcarries deletions of PDC1 and PDC5 with the concomitant expression ofthe truncated version of the MTH1 and the secondary alcoholdehydrogenase of C. beijerinckii of the 2-propanol pathway.

Strain BRKY-163 is the PDC-null strain with the concomitant expressionof the truncated version of the MTH1 gene and the secondary alcoholdehydrogenase of C. beijerinckii. This strain was obtained as theprogeny of a cross between BRKY-130 and BRKY-138 with the tetrads beingscreened by PCR for deletions in both PDC1 and PDC6 loci, since both thePDC5 deletion (containing the truncated MTH1 gene) and the secondaryalcohol dehydrogenase integration were inherited from both parents andas such were not subject to segregation.

Strain BRKY-174 is a PDC-null strain with the concomitant expression ofthe truncated version of the MTH1 gene, the secondary alcoholdehydrogenase of C. beijerinckii, and the two subunits of the acetyl-CoAacetoacetate CoA transferase of E. coli. Strain BRKY-174 was obtained byintegrating at locus Chr X: 194944..195980 of strain BRKY-163 aconstruct for the expression of the gene atoA from E. coli controlled bythe TEF1 promoter and CYC1 terminator and the atoD gene from E. colicontrolled by the PGK1 promoter and ADH1 terminator, which code for thetwo subunits of the acetyl-CoA acetoacetate CoA transferase from E. coli(Enzyme No. C1, E.C. Number 2.8.3.8, Table 6). The integration cassettewas flanked by ˜150 bp homology regions for locus Chr X: 194944..195980and comprised the TRP1 auxotrophic marker for strain selection and wasbuilt by overlapping PCR (SEQ ID NO:8). Upon introduction in a S.cerevisiae host cell, this construct can integrate by homologousrecombination into the locus Chr X: 194944..195980 locus of the genome.The resulting strain was selected for triptophan prototrophy in YeastNitrogen Base Media without tryptophan (Sigma) and confirmed by PCRamplification and sequencing of genomic DNA.

Strain BRKY-189 is a PDC-null strain with the concomitant expression ofthe truncated version of the MTH1 gene, the secondary alcoholdehydrogenase of C. beijerinckii, the two subunits of the acetyl-CoAacetoacetate CoA transferase from E. coli, the thiolase gene fromClostridium acetobutylicum, and the acetoacetate decarboxylase gene fromC. beijerinckii, thus expressing a full 2-propanol producing pathwayfrom acetyl-CoA. Strain BRKY-189 was obtained by integrating at locusYPRCtau3 of strain BRKY-174 a construct for the expression of thethiolase gene from C. acetobutylicum (Enzyme No. B, E.C. number 2.3.1.9,Table 6) controlled by the ADH1 promoter and TEF1 terminator and theacetoacetate decarboxylase gene from C. beijerinckii (Enzyme No. D, E.C.number 4.1.1.4, Table 6) controlled by the TDH3 promoter and TRP1terminator. The integration cassette was flanked by ˜150 bp homologyregions for locus YPRCtau3 and comprised the Nourseotricin marker forstrain selection and was bilt by overlapping PCR (SEQ ID NO:9). Uponintroduction in a S. cerevisiae host cell, this construct can integrateby homologous recombination into the YPRCtau3 locus of the genome. Theresulting strain was selected for nourseotricin resistance in YPD Mediasupplemented with 100 micrograms per mililiter of nourseotricin andconfirmed by PCR amplification and sequencing of genomic DNA.

Strain BRKY-272 was obtained by transforming strain BRKY-189 with asingle copy plasmid (pRS415-LEU backbone, ATCC® 87520™) expressing thefollowing genes and control sequences: 1) PFLA gene from E. colicontrolled by the PGK1 promoter and ADH1 terminator, 2) PFLB gene fromE. coli controlled by the TEF1 promoter and TDH3 terminator, and 3) udhAgene from E. coli controlled by the TDH3 promoter and ADH1 terminator.The resulting plasmid sequence is included as SEQ ID NO:10. Theresulting strain thus comprises a full pathway for the conversion of thecytosolic pyruvate intermediate into cytosolic acetyl-CoA in addition tothe full pathway for the conversion of the acetyl-CoA intermediate into2-propanol integrated in the genome of the BRKY-189 parent strain.Strain BRKY-272 is thus a 2-propanol and ethanol-null production strainable to produce cytosolic acetyl-CoA.

Strain BRKY-397 was obtained transforming strain BRKY-189 with a singlecopy plasmid (pRS415-LEU backbone, ATCC® 87520™) that cofers prototrophyto leucine and was used as a control strain.

TABLE 8 Oligonucleotides used to build strains byhomologous recombination Name DNA Sequence (5′→3′) BKO592ACTCATAACCTCACGCAAAATAACACAGTCAAATCAATCAAAAGCTTTTCAATTCAATTCATC (SEQ ID NO: 108) BKO593AATGCTTATAAAACTTTAACTAATAATTAGAGATTAAATCGCGGGTAATAACTGATATAAT (SEQ ID NO: 109) BKO678CCTAGATCGATTTGATTACAGGATAAGGGATATGGTGCGATTCGCGAGCTTTTACCAATATGTATAAAAGGCGGCTGTTTGAAGCCATTCTATCTTAATCTTGTGCTATTGCAGTCCTCTTTTATATACAGTATAAATAAAAAACCCACGTAATATAGCAAAAACATATTGCCAACAAAAGCTTTTCAATTCAATTCATC (SEQ ID NO: 110) BKO679GGGTAATAACTGATATAATGCCATTAGTAGTGTACTCAAACGAATTATTGTTGCAAATAAATAAACTTACACAGTTTGAATACATAAATCAATCAGACAAATAAATACATCGGTTCAAATTATACTAAATCTAAATACTACGTTATCGCCGTGAATTACGCAATTCGCATGTTACGTACTGCGCGTCTCTTGTTGAATA (SEQ ID NO: 111) BKO909TCAAGTTCCTCGATACTAGTTATTTGTAATACGTATACGAATTCCTTCAACAAAGGCCAAGGAAATAAAGCAAATAACAATAACACCATTATTTTAATTTTTTTTCTATTACTGTCGCTAACACCTGTATGGTTGCAACCAGGTGAGAATCCTTCTGATGCATACTTTATGCGTTTATGCTACGCTGCAGGTCGACAA (SEQ ID NO: 112) BKO910GCTAAAGGTACAAAACCGAATACGAAAGTAAATAAATTAATCAGCATAAAATTAAATAATAAACCACCTAAAATATTAGAAGCTAATCTTTAACCTGGAAGACAGGACAGAAAAGTAATTACAAGAACATATGTGAAAAAAAATAGTTGATATTTTAAACCAAATCAGAAATTTATTATACAGAGCGCCCAATACGCAAA (SEQ ID NO: 113)

By providing a PDC-null strain with 1) a truncated MTH1 gene toalleviate 2C auxotrophy and increase glucose tolerance (Oud et al.Microbial Cell Factories, vol. 11, 2012, p. 131-140); 2) a pathway forthe production of cytosolic acetyl-CoA from pyruvate (Pyruvate FormateLyase and PFL-activating enzyme coding genes from E. coli) that is onlyfunctional under anaerobic conditions; and 3) a temporary redox sink(udhA transhydrogenase coding gene from E. coli) that would enablereoxidation of the excess NADH produced at the end of glycolysis, thestrain should be able to grow under anaerobic conditions. This would bedifferent from parental strain BRKY-189 that lacks a cytosolicacetyl-CoA production pathway and is redox imbalanced under anaerobicconditions, and, thus, it is not expected to grow under strict anaerobicconditions.

To test this hypothesis, strains BRKY-397 and BRKY-272 were cultured inYNB Media without leucine (to select for the pRS415 plasmid) containing8 g/L of glucose as the sole carbon source. The full composition of themedia follows: Glucose, 8 g/L, Ammonium sulfate, 5.0 g/L, Biotin, 2.0micrograms/L, Calcium pantothenate, 400 micrograms/L, Folic acid, 2.0micrograms/L, Inositol, 2.0 mg/L, Nicotinic acid, 400 micrograms/L,p-Aminobenzoic acid, 200 micrograms/L, Pyridoxine HCl, 400 micrograms/L,Riboflavin, 200 micrograms/L, Thiamine HCL, 400 micrograms/L, Citricacid, 0.1 g/L, Boric acid, 500 micrograms/L, Copper sulfate, 40micrograms/L, Potassium iodide, 100 micrograms/L, Ferric chloride, 200micrograms/L, Magnesium sulfate, 400 micrograms/L, Sodium molybdate, 200micrograms/L, Zinc sulfate, 400 micrograms/L, Potassium phosphatemonobasic, 1.0 g/L, Magnesium sulfate, 0.5 g/L, Sodium chloride, 0.1g/L, Calcium chloride, 0.1 g/L, all standard amino acids except forleucine at a concentration of 76 mg/L, Adenine, 18 mg/L, inositol, 76mg/L, p-aminobenzoic acid (8 mg/L), uracil (76 mg/L). All reagents formedia preparation were obtained from Sigma (YNB without amino acids,Part No. Y0626, Yeast Synthetic Drop-out Medium Supplements withoutleucine, Part No. Y1376).

The strains were first grown in aerobic shake flasks for 24 hours untilreaching an OD₆₀₀=˜4. Cells from this aerobic pre-culture wereinoculated to an OD600 of ˜0.3 in sealed shake flasks purged with N₂ gasfor 20 minutes before and immediately after inoculation. To preventpositive pressure build-up from CO₂ production, the flasks were providedwith an off-gas tube that bubbled into a glass bottle containingsterilized water. The strains were cultured for up to 300 h. Sampleswere taken daily through a built-in sampling syringe. FIG. 5 shows aschematic representation of the anaerobic flask system.

Cell biomass was calculated by measuring the absorbance at 600 nm in aULTROSPEC 2000 spectrophotometer UV/visible (Pharmacia Biotech) afterappropriate dilution in saline. For HPLC-RI analysis, the samples werefiltered through a 0.2 μm filter (Millipore). pyruvic, lactic and aceticacids, ethanol, glycerol, 2-propanol, 1,2-propanediol and sugars wereseparated and quantified by high-performance liquid chromatography(Waters 600 Chromatograph), using an ion exclusion column Aminex HPX-87H(Bio-Rad). Operating conditions were: 0.04 mol L⁻¹ H₂SO₄ degassedeluent, flow rate 0.6 mL min⁻¹, column temperature 35° C. andrefractometer temperature 35° C.

FIG. 6A shows increased growth of the BRKY-272 strain compared to thecontrol (BRKY-397) in the anaerobic flask system shown in FIG. 5. Themarginal growth of the control strain (BRKY-397) is hypothetized to haveresulted from traces of oxygen present in the anaerobic flask system.However, we show that the growth of strain BRKY-272 is significantlyincreased compared to the control, as well as the consumption of glucoseand production of metabolites as shown in FIGS. 6B (control strainBRKY-397) and FIG. 6C (test strain BRKY-272). None of the strainsproduced ethanol, thus confirming the PDC-null genotype. The resultsshown in FIG. 6 are representative of at least three independentreplicates. Although strain BRKY-272 contains all genes in a pathway forthe convertion of the acetyl-CoA intermediate into 2-propanol, theproduction of 2-propanol was not expected due to the absence of acetatethat would act as an acceptor for the acetoacetyl-CoA:acetate CoAtransferase (atoAD from E. coli) or a Acetoacetyl-CoA acyl-CoA thiolase.

Example 1 thus shows that it was possible to restore anaerobic growth aPDC-null yeast strain by providing three key elements: 1) a truncatedMTH1 gene to alleviate 2C auxotrophy and increase glucose tolerance (Oudet al. Microbial Cell Factories, vol. 11, 2012, p. 131-140); 2) apathway for the production of cytosolic acetyl-CoA from pyruvate(Pyruvate Formate Lyase and PFL-activating enzyme coding genes from E.coli) that is only functional under anaerobic conditions; and 3) atemporary redox sink (udhA transhydrogenase coding gene from E. coli)that would enable reoxidation of the excess NADH produced at the end ofglycolysis.

Example 2 Modification of a Microorganism for Production of1,2-Propanediol and 2-Propanol Without Production of Ethanol

This example demonstrates the construction of yeast strain BRKY-399(haploid and isogenic to Saccharomyces cerevisiae S288C) simultaneouslyexpressing genes coding for enzymes in a pathway that catalyze theconversion of an acetyl-CoA intermediate to 2-propanol and adihydroxyacetone intermediate to 1,2-propanediol. The strain furthercomprises deletions of the PDC1, PDC5 and PDC6 genes coding for thethree pyruvate decarboxylase isoforms, and thus lacks pyruvatedecarboxylase activity. The strain further comprises an integration of agene expressing the truncated version of the MTH1 enzyme as set forth inSEQ ID NO: 2.

The strains listed in Table 9 represent the step-wise creation of strainBRKY-399. All DNA-mediated transformation into S. cerevisiae wasconducted using the Lithium Acetate procedure as described by Gietz R Wand Woods R A, Guide to Yeast Gentics and Molecular Cell Biology. PartB. San Diego, Calif.: Academic Press Inc. pp. 87-96 (2002) and in allcases integration of the constructs was confirmed by PCR amplificationof genomic DNA. In some cases, strains with more than one desired traitwere obtained by crossing haploid strains of compatible mating types. Inthese cases, diploid construction, sporulation, tetrad dissection, andrandom spore analysis was performed according to Treco D A and WinstonF, UNIT 13.2 Growth and Manipulation of Yeast, Curr. Protoc. Mol. Biol.82:13.2.1-13.2.12 (2008).

TABLE 9 Strains representing the step-wise creation of strain BRKY-399that simultaneously expresses all genes coding for enzymes in a pathwaythat catalyze the conversion of an acetyl-CoA intermediate to 2-propanoland a dihydroxyacetone intermediate to 1,2-propanediol and furthercomprises deletions of the PDC1, PDC5 and PDC6 genes coding for thethree pyruvate decarboxylase isoforms and the integration of a genecoding for the truncated version of the MTH1 gene Strain ID StrainGenotype Vectors Host FY23 S288C, mating type a, ura3-52, leu2Δ1,trp1Δ63 none N/A FY86 S288C, mating type alpha, ura3-52, leu2Δ1, noneN/A his3delta200 BRKY-02 S288C, mating type a, ura3-52, leu2Δ1, trp1Δ63,none FY23 PDC1::URA3 BRKY-31 S288C, mating type a; his3delta200,ura3-52, none FY23 × FY86 trp1delta63, leu2delta1 progeny BRKY-37 S288C,mating type alpha; his3delta200, ura3-52, none FY86 × BRKY02trp1delta63, leu2delta1, PDC1::URA3 progeny BRKY-69 S288C, mating typea; his3delta200, ura3-52, none BRKY31 trp1delta63, leu2delta1,PDC6::URA3 BRKY-86 S288C, mating type a; his3delta200, ura3-52, noneBRKY69 trp1delta63, leu2delta1, PDC6::URA3, locus Chr XI: 91575-adh/HIS3BRKY-97 S288C, mating type a; his3delta200, ura3-52, none BRKY31trp1delta63, leu2delta1, PDC5:KanMX4_tMTH1 BRKY-115 S288c, mating typeα; his3delta200, ura3-52, none BRKY37 × BRKY97 trp1delta63, leu2delta1,PDC5:KanMX4_tMTH1 progeny BRKY-118 S288c, mating type alpha;his3delta200, ura3-52, none BRKY37 × BRKY97 trp1delta63, leu2delta1,PDC5:KanMX4_tMTH1 progeny PDC1::URA3 BRKY-130 S288C, mating type α;his3delta200, ura3-52, none BRKY115 × BRKY86 trp1delta63, leu2delta1,PDC5::KanMx4/MTH1, progeny PDC6::URA3, locus Chr XI: 91575-adh/HIS3BRKY-138 S288C, mating type a; his3delta200, ura3-52, none BRKY86 ×BRKY118 trp1delta63, leu2delta1, PDC1::URA3, progeny PDC5::KanMX4/MTH1,locus Chr XI: 91575- adh/HIS3 BRKY-163 S288C, mating type alpha;his3delta200, ura3-52, none BRKY138 × BRKY130 trp1delta63, leu2delta1,PDC1::URA3, progeny PDC5::KanMX4/MTH1, PDC6::URA3 locus Chr XI:91575-adh/HIS3 BRKY-174 S288C, mating type alpha; his3delta200, ura3-52,none BRKY163 trp1delta63, leu2delta1, PDC1::URA3, PDC5::KanMX4/MTH1,PDC6::URA3, locus Chr XI: 91575-adh/HIS3, locus ChrX: 194944-atoA/atoD/TRP1 BRKY-189 S288C, mating type alpha; his3delta200, ura3-52,none BRKY174 trp1delta63, leu2delta1, PDC1::URA3, PDC5::KanMX4/MTH1,PDC6::URA3, locus Chr XI: 91575-adh/HIS3, ChrX: 194944- atoA/atoD/TRP1,locus YPRCtau3::thl/adc/natMX BRKY 399 S288C, mating type alpha;his3delta200, ura3-52, pRS415−LEU− BRKY189 trp1delta63, leu2delta1,PDC1::URA3, PGK1+yqhd*+tADH1, PDC5::KanMX4/MTH1, PDC6::URA3, locus ChrpPGK1+Gre2*+tCYC1, XI: 91575-adh/HIS3, ChrX: 194944-pTPI1+mgsA−Bs+tTDH3, atoA/atoD/TRP1, locus YPRCtau3::thl/adc/natMX;pTPI1+mgsA−Bs+tTDH3, LEU2, pPGK1+yqhd*+tADH1, pTPI1+mgsA−Bs+tTDH3,pPGK1+Gre2*+tCYC1, pTPI1+mgsA−Bs+tTDH3, pTEF1+udhA_Ec_tTDH3pTPI1+mgsA−Bs+tTDH3, pTPI1+mgsA−Bs+tTDH3, pTEF1+udhA_ Ec_tTDH3

FY23 and FY86 are haploid strains isogenic to Saccharomyces cerevisiaeS288C as described by Winston et al. 1995, Yeast, 11, issue 1, 53-55),each containing three auxotrophic markers, and were used as the“wild-type” strains for this study.

Strain BRKY-02 was obtained by deleting the PDC1 gene from strain FY23with a URA3 marker in a linear construct. The linear construct was builtby PCR amplification of the URA3 marker gene from the commercial vectorpESC-URA with primers BK0592 and BKO593 (Table 8) containing 40 bp 5′extensions corresponding to regions upstream and downstream of the PDC1locus (SEQ ID NO: 4). Upon introduction in a S. cerevisiae host cell,this construct can integrated by homologous recombination into the PDC1locus of the genome, functionally disrupting PDC1p by replacing thePDC1p coding sequence with its integrating sequence. The resultingstrain was selected for uracyl prototrophy in Yeast Nitrogen Base Mediawithout uracyl (Sigma) and confirmed by PCR amplification of genomicDNA.

Strains BRKY-31 and BRKY-37 were generated by crossing and tetraddissection of strains FY23xFY86 and FY86xBRKY-02, respectively. Theobjective was to obtain haploid strains with four auxotrophic markersand, in the case of BRKY-37, the four auxotrophic markers and thePDC1::URA3 deletion.

Strain BRKY-69 was obtained by deleting the PDC6 gene from strainBRKY-31 with a URA3 marker in a linear construct. The linear constructwas built by PCR amplification of the URA3 marker gene from thecommercial vector pESC-URA with primers BK0678 and BK0679 (Table 8)containing 40 bp 5′ extensions corresponding to regions upstream anddownstream of the PDC6 locus (SEQ ID NO:5). Upon introduction in a S.cerevisiae host cell, this construct can integrate by homologousrecombination into the PDC6 locus of the genome, functionally disruptingPDC6p by replacing the PDC6p coding sequence with its integratingsequence. The resulting strain was selected for uracyl prototrophy inYeast Nitrogen Base Media without uracyl (Sigma) and confirmed by PCRamplification of genomic DNA.

Strain BRKY-86 was obtained by integrating at locus Chr XI:91575-..92913of strain BRKY-69 a construct for the expression of the secondaryalcohol dehydrogenase from Clostridium beijerinckii (Table 6, Enzyme No.E, E.C. Number 1.1.1.2) controlled by the TEF1 promoter and the PGK1terminator. The integration cassette was flanked by ˜150 bp homologyregions for locus Chr XI: 91575..92913 and comprised the HIS3auxotrophic marker for strain selection. The whole construct was builtby overlapping PCR (SEQ ID NO:6). Upon introduction in a S. cerevisiaehost cell, this construct can integrate by homologous recombination intothe Chr XI:91575-..92913 locus of the genome. The resulting strain wasselected for histidine prototrophy in Yeast Nitrogen Base Media withouthistidine (Sigma) and confirmed by PCR amplification and sequencing ofgenomic DNA.

Strain BKRY-97 was obtained by deleting the PDC5 gene from strainBRKY-31 with a linear construct comprising the KanMX4 marker gene and aexpression cassette for the truncated version of the MTH1 enzyme setforth in SEQ ID NO: 2 flanked by homology sequences consisting of theupstream and downstream nucleotide sequences of the S. cerevisiae PDC5locus. A truncated version of the MTH1 gene was synthesized by IDT(Integrated DNA Technologies, Coralville, Iowa). Next, the truncatedMTH1 gene was introduced into a surrogate plasmid vector carrying theKanMX4 selection marker flanked by loxp recognition sequences (pUG6vector by Euroscarf). PCR amplification of the truncated MTH1 gene andKanMX4 marker gene from the surrogate vector was performed with primersBKO909 and BKO910 (Table 8) containing 5′ extensions corresponding toregions upstream and downstream of the PDC5 locus (SEQ ID NO:7). Uponintroduction in a S. cerevisiae host cell, this construct can integrateby homologous recombination into the PDC5 locus of the genome,functionally disrupting PDC5p by replacing the PDC5p coding sequencewith its integrating sequence for the concomitant expression of thetruncated version of MTH1 and KanMX4. The resulting strain was selectedfor Geneticin resistance in YPD Media containing 150 micrograms perliter of Geneticin and confirmed by PCR amplification and sequencing ofgenomic DNA.

Strain BRKY-115 is a progeny of a cross between strains BRKY-37 andBRKY-97 and differs from its parent BRKY-97 only in the mating type.This different mating type was used in a further cross aimed atobtaining the PDC-null triple deletion strain.

Strain BRKY-118 is an ethanol-null strain carrying deletions in genesPDC1 and PDC5, coding for the two most important isoforms of pyruvatedecarboxylases. Deletion of both genes causes significantly decreasedamounts of ethanol from sugar and creates a C2-auxotrophy. Aftersequential deletion of the two genes failed repeatedly, we performed across between strains BRKY-37 and BRKY-97 and selected tetrads on thebasis of uracyl prototrophy and Geneticin resistance and were able toobtain the PDC1/PDC5 double knockout.

Strain BRKY-130 is the progeny of a cross between BRKY115×BRKY86 andcarries deletions of PDC6 and PDC5 with the concomintant expression ofthe truncated version of the MTH1 and the secondary alcoholdehydrogenase of C. beijerinckii of the 2-propanol pathway.

Strain BRKY-138 is the progeny of a cross between BRKY86 and BRKY118 andcarries deletions of PDC1 and PDC5 with the concomitant expression ofthe truncated version of the MTH1 and the secondary alcoholdehydrogenase of C. beijerinckii of the 2-propanol pathway.

Strain BRKY-163 is the PDC-null strain with the concomitant expressionof the truncated version of the MTH1 gene and the secondary alcoholdehydrogenase of C. beijerinckii. This strain was obtained as theprogeny of a cross between BRKY-130 and BRKY-138 with the tetrads beingscreened by PCR for deletions in both PDC1 and PDC6 loci, since both thePDC5 deletion (containing the truncated MTH1 gene) and the secondaryalcohol dehydrogenase integration were inherited from both parents andas such were not subject to segregation.

Strain BRKY-174 is a PDC-null strain with the concomitant expression ofthe truncated version of the MTH1 gene, the secondary alcoholdehydrogenase of C. beijerinckii, and the two subunits of the acetyl-CoAacetoacetate CoA transferase of E. coli. Strain BRKY-174 was obtained byintegrating at locus Chr X: 194944..195980 of strain BRKY-163 aconstruct for the expression of the gene atoA from E. coli controlled bythe TEF1 promoter and CYC1 terminator and the atoD gene from E. colicontrolled by the PGK1 promoter and ADH1 terminator, which code for thetwo subunits of the acetyl-CoA acetoacetate CoA transferase from E. coli(Enzyme No. C1, E.C. Number 2.8.3.8, Table 6). The integration cassettewas flanked by ˜150 bp homology regions for locus Chr X: 194944..195980and comprised the TRP1 auxotrophic marker for strain selection and wasbuilt by overlapping PCR (SEQ ID NO:8). Upon introduction in a S.cerevisiae host cell, this construct can integrate by homologousrecombination into the locus Chr X: 194944..195980 locus of the genome.The resulting strain was selected for triptophan prototrophy in YeastNitrogen Base Media without tryptophan (Sigma) and confirmed by PCRamplification and sequencing of genomic DNA.

Strain BRKY-189 is a PDC-null strain with the concomitant expression ofthe truncated version of the MTH1 gene, the secondary alcoholdehydrogenase of C. beijerinckii, the two subunits of the acetyl-CoAacetoacetate CoA transferase from E. coli, the thiolase gene fromClostridium acetobutylicum, and the acetoacetate decarboxylase gene fromC. beijerinckii, thus expressing a full 2-propanol producing pathwayfrom acetyl-CoA. Strain BRKY-189 was obtained by integrating at locusYPRCtau3 of strain BRKY-174 a construct for the expression of thethiolase gene from C. acetobutylicum (Enzyme No. B, E.C. number 2.3.1.9,Table 6) controlled by the ADH1 promoter and TEF1 terminator and theacetoacetate decarboxylase gene from C. beijerinckii (Enzyme No. D, E.C.number 4.1.1.4, Table 6) controlled by the TDH3 promoter and TRP1terminator. The integration cassette was flanked by ˜150 bp homologyregions for locus YPRCtau3 and comprised the Nourseotricin marker forstrain selection and was bilt by overlapping PCR (SEQ ID NO:9). Uponintroduction in a S. cerevisiae host cell, this construct can integrateby homologous recombination into the YPRCtau3 locus of the genome. Theresulting strain was selected for nourseotricin resistance in YPD Mediasupplemented with 100 micrograms per mililiter of nourseotricin andconfirmed by PCR amplification and sequencing of genomic DNA.

Strain BRKY-397 was obtained transforming strain BRKY-189 with a singlecopy plasmid (pRS415-LEU backbone, ATCC® 87520™) that cofers prototrophyto leucine and was used as a control strain.

Strain BRKY-399 was obtained by transforming strain BRKY-189 with asingle copy plasmid (pRS415-LEU backbone, ATCC® 87520™) expressing thefollowing genes: 1) three copies of the Bacillus subtilis mgsA (EnzymeNo. F1, E.C. number 4.2.3.3), each controlled by the TPI1 promoter andthe TDH3 terminator, 2) one copy of the yqhD gene from E. colicontrolled by the PGK1 promoter and ADH1 terminator, 3) one copy of theGRE2 gene from S. cerevisiae controlled by the PGK1 promoter and CYC1terminator, and 4) one copy of the udhA gene from E. coli controlled bythe TDH3 promoter and ADH1 terminator. The resulting plasmid sequence isprovided in SEQ ID NO:11. The resulting strain BRKY-399 thus comprises afull pathway for the conversion of the dihydroxyacetone-phosphateintermediate into 1,2-propanediol in addition to the full pathway forthe conversion of the acetyl-CoA intermediate into 2-propanol integratedin the genome of the BRKY-189 parent strain. Strain BRKY-399 is thus anethanol-null 2-propanol and 1,2-propanediol co-production strain.

Example 3 Fermentation of Glucose by Genetically Modified Ethanol-NullMicroorganism to Produce 1,2-Propanediol and 2-Propanol

In this example, a genetically modified yeast strain BRKY-399, asproduced in Example 2 above, was used to ferment a C6 sugar as a solecarbon source (glucose) and a C2 carbon source (sodium acetate) toco-produce 1,2-propanediol and 2-propanol in a two phase culture systemin bioreactors. Since strain BRKY-399 lacks a functional pathway for theproduction of the acetyl-CoA intermediate from the pyruvate intermediate(i.e. PFL enzyme from E. coli), the culture media must be supplied withpotassium acetate to serve as a substrate for acetyl-CoA formation fromeither the acetyl-CoA synthase or the acetoacetyl-CoA: acetate CoAtransferase.

Strain BRKY-399 was cultured in YNB Media without leucine (to select forthe pRS415 plasmid) containing 8 g/L of glucose and 0.5 g/L sodiumacetate as the carbon sources. The full composition of the mediafollows: Glucose, 8 g/L, Sodium acetate, 0.5 g/L, Ammonium sulfate, 5.0g/L, Biotin, 2.0 micrograms/L, Calcium pantothenate, 400 micrograms/L,Folic acid, 2.0 micrograms/L, Inositol, 2.0 mg/L, Nicotinic acid, 400micrograms/L, p-Aminobenzoic acid, 200 micrograms/L, Pyridoxine HCl, 400micrograms/L, Riboflavin, 200 micrograms/L, Thiamine HCL, 400micrograms/L, Citric acid, 0.1 g/L, Boric acid, 500 micrograms/L, Coppersulfate, 40 micrograms/L, Potassium iodide, 100 micrograms/L, Ferricchloride, 200 micrograms/L, Magnesium sulfate, 400 micrograms/L, Sodiummolybdate, 200 micrograms/L, Zinc sulfate, 400 micrograms/L, Potassiumphosphate monobasic, 1.0 g/L, Magnesium sulfate, 0.5 g/L, Sodiumchloride, 0.1 g/L, Calcium chloride, 0.1 g/L, all standard amino acidsexcept for leucine at a concentration of 76 mg/L, Adenine, 18 mg/L,inositol, 76 mg/L, p-aminobenzoic acid (8 mg/L), uracil (76 mg/L). Allreagents for media preparation were obtained from Sigma (YNB withoutamino acids, Part No. Y0626, Yeast Synthetic Drop-out Medium Supplementswithout leucine, Part No. Y1376).

Free-cell batch fermentation was conducted in a 0.6 L bioreactor(Multifors—Infors) containing 0.4 L of the sterile medium inoculated atan initioal OD600 of ˜0.3 with freshly harvested cells of strainBRKY-399 grown in aerobic pre-culture. The bioreactor temperature wasmaintained at 30° C. The fermentation was conducted in two phases: onephase for aerobic production of biomass and a second microaerobic phasefor product formation (synthetic air was supplied in the headspace butnot sparged in the medium). During the first phase, aerobic conditionswere maintained by sparging with synthetic air at a rate of 0.1 L/minand agitation speed of 150 rpm. Initial pH was 5.8 and was allowed todrop to a level of 3.5 and then maintained at 3.5 by addingautomatically a 1 M NaOH solution. Once glucose and acetate wereexhausted and the OD600 reached a value >10 (˜48 h), a second pulse of 8g/L glucose and 0.2 g/L acetate was injected in the bioreactor and thesynthetic air sparging shifted to headspace and the agitation speed wasincreased to 450 rpm. This second phase was allowed to continue for ˜160h. Potassium acetate was supplied at a concentration of 0.2 g/L wheneverneeded.

Sampling was performed daily. Cell biomass was calculated by measuringthe absorbance at 600 nm in a ULTROSPEC 2000 spectrophotometerUV/visible (Pharmacia Biotech) after appropriate dilution in saline. ForHPLC-RI and HPLC-UV analyses, the samples were filtered through a 0.2 μmfilter (Millipore). Pyruvic, lactic and acetic acids, ethanol, glycerol,2-propanol, 1,2-propanediol and sugars were separated and quantified byhigh-performance liquid chromatography (Waters 600 Chromatograph), usingan ion exclusion column Aminex HPX-87H (Bio-Rad) and the IR and UVdetectors in series. Operating conditions were: 0.04 mol L⁻¹ H₂SO₄degassed eluent, flow rate 0.6 mL min⁻¹, column temperature 35° C. andrefractometer temperature 50° C.

FIG. 7 show an exemplary carbon source consumption and metaboliteprofile of the two-phase fermentation of strain BRKY-399. Under theconditions provided it was possible to co-produce 2-propanol (200 mg/L)and 1,2-Propanediol (30 mg/L) after 160 h of culture from Glucose andsodium acetate (Total product yield=2.45 g per 100 g of substratesconsumed). Three independent experiments were conducted and yieldedsimilar results with regards to total product yield, although therelative amounts of 2-propanol and 1,2-PDO varied according to thespecific microaerobic conditions.

This example shows that it is possible to use the ethanol-null yeastchassis for the co-production of bulk chemicals.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein can be further limited in theclaims using consisting of or and consisting essentially of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosedherein are illustrative of the principles of the present disclosure.Other modifications that can be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure can be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of materials and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary, only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

1. A non-naturally occurring microorganism comprising: a disruption ofone or more enzymes that decarboxylate pyruvate and/or a disruption ofone or more transcription factors of one or more enzymes thatdecarboxylate pyruvate; a genetic modification that substantiallydecreases glucose import into the microorganism; one or morepolynucleotides encoding one or more enzymes in a pathway that producescytosolic acetyl-CoA; one or more polynucleotides encoding one or moreenzymes in a pathway that catalyze a conversion of cytosolic acetyl-CoAto 2-propanol; and one or more polynucleotides encoding one or moreenzymes in a pathway that catalyze a conversion ofdihydroxyacetone-phosphate to 1-propanol and/or 1,2-propanediol.)
 2. Thenon-naturally occurring microorganism of claim 1, wherein the disruptionin the one or more enzymes that decarboxylate pyruvate is a deletion ora mutation.)
 3. The non-naturally occurring microorganism of claim 1,wherein the one or more enzymes that decarboxylate pyruvate includepdc1, pdc 5, and/or pdc6, and wherein the one or more transcriptionfactors of the one or more enzymes that decarboxylate pyruvate includepdc2.)
 4. The non-naturally occurring microorganism of claim 1, whereinthe microorganism comprises an exogenous polynucleotide that encodes atranscription factor involved in glucose import.)
 5. The non-naturallyoccurring microorganism of claim 1, wherein the microorganism comprisesa genetic modification in an endogenous polynucleotide that encodes atranscription factor involved in glucose import.)
 6. The non-naturallyoccurring microorganism of claim 5, wherein the genetic modification isa truncation of the MTH1 transcription factor.)
 7. The non-naturallyoccurring microorganism of claim 1, wherein the one or more exogenouspolynucleotides encoding one or more enzymes in a pathway that producesacetyl-CoA encode i.) pyruvate formate lyase and pyruvate formate lyaseactivating enzyme, ii) pyruvate dehydrogenase, dihydrolipoyltransacetylase and dihydrolipoamide dehydrogenase, iii) pyruvatedehydrogenase, dihydrolipoyl transacetylase, dihydrolipoamidedehydrogenase, and pyruvate dehydrogenase complex protein X, or anycombination thereof.)
 8. The non-naturally occurring microorganism ofclaim 1, wherein the microorganism is a eukaryote selected from thegroup consisting of: yeast, filamentous fungi, protozoa, and algae.) 9.The non-naturally occurring microorganism of claim 1, wherein the one ormore polynucleotides coding for enzymes in a pathway that catalyzes aconversion of acetyl-CoA to 2-propanol include: one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetyl-CoA to acetoacetyl-CoA, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion ofacetoacetyl-CoA to acetoacetate, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of acetoacetate toacetone, and/or one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of acetone to 2-propanol.)
 10. Thenon-naturally occurring microorganism of claim 1, wherein the one ormore polynucleotides coding for enzymes in a pathway that catalyzes aconversion of dihydroxyacetone-phosphate to 1-propanol and/or1,2-propanediol include: one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol.)
 11. A non-naturally occurring microorganism comprising: oneor more exogenous polynucleotides encoding one or more enzymes in apathway that produces cytosolic acetyl-CoA; one or more polynucleotidescoding for enzymes that catalyze a conversion of cytosolic acetyl-CoA to2-propanol; and one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of dihydroxyacetone-phosphate to1-propanol and/or 1,2-propanediol, wherein the microorganism has reducedlevels of pyruvate decarboxylase enzymatic activity, and wherein themicroorganism is capable of growing on a C6 sugar as a sole carbonsource under anaerobic conditions.)
 12. The non-naturally occurringmicroorganism of claim 11, wherein the microorganism has a disruption inone or more polynucleotides that code for one or more enzymes thatdecarboxylate pyruvate or a disruption in one or more polynucleotidesthat code for a transcription factor of an enzyme that decarboxylatespyruvate.)
 13. The non-naturally occurring microorganism of claim 11,wherein the disruption in the one or more enzymes that decarboxylatepyruvate is a deletion or a mutation.)
 14. The non-naturally occurringmicroorganism of claim 13, wherein the one or more enzymes thatdecarboxylate pyruvate include pdc1, pdc 5, and/or pdc6, and wherein theone or more transcription factors of the one or more enzymes thatdecarboxylate pyruvate include pdc2.)
 15. The non-naturally occurringmicroorganism of claim 11, wherein the one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofacetyl-CoA to 2-propanol include: one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of acetyl-CoA toacetoacetyl-CoA, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of acetoacetyl-CoA to acetoacetate,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol.)
 16. The non-naturally occurringmicroorganism of claim 11, wherein the one or more polynucleotidescoding for enzymes in a pathway that catalyzes a conversion ofdihydroxyacetone-phosphate to 1-propanol include and/or 1,2-propanediol:one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of methylglyoxal to hydroxyacetone, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol.)
 17. A method for co-producing 2-propanol with 1-propanoland/or 1,2-propanediol from a fermentable carbon source under anaerobicconditions, the method comprising: a.) providing a fermentable carbonsource; b.) contacting the fermentable carbon source with thenon-naturally occurring microorganism of claim 1 in a fermentation mediaunder substantially anaerobic conditions, and c.) expressing thepolynucleotides in the microorganism for the co-production of 2-propanolwith 1-propanol and/or 1,2-propanediol, wherein the microorganismco-produces 2-propanol with 1-propanol and/or 1,2-propanediol.)
 18. Themethod of claim 17, wherein the fermentable carbon source is sugarcanejuice, sugarcane molasses, hydrolyzed starch, hydrolyzed lignocellulosicmaterials, glucose, sucrose, fructose, lactate, lactose, xylose,pyruvate, or glycerol in any form or mixture thereof.)
 19. The method ofclaim 17, wherein the fermentable carbon source is a monosaccharide,oligosaccharide, or polysaccharide.)
 20. The method of claim 17, whereinthe produced 2-propanol with 1-propanol and/or 1,2-propanediol aresecreted by the microorganism into the fermentation media.)
 21. Themethod of claim 20 further comprising recovering the produced 2-propanolwith 1-propanol and/or 1,2-propanediol from the fermentation media.